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Citation: Naryzhny, S.; Ronzhina, N.;
Zorina, E.; Kabachenko, F.; Klopov,
N.; Zgoda, V. Construction of 2DE
Patterns of Plasma Proteins: Aspect
of Potential Tumor Markers. Int. J.
Mol. Sci. 2022,23, 11113. https://
doi.org/10.3390/ijms231911113
Academic Editor: Peter J.K. Kuppen
Received: 15 August 2022
Accepted: 16 September 2022
Published: 21 September 2022
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International Journal of
Molecular Sciences
Article
Construction of 2DE Patterns of Plasma Proteins: Aspect of
Potential Tumor Markers
Stanislav Naryzhny 1, 2, * , Natalia Ronzhina 2, Elena Zorina 1, Fedor Kabachenko 3, Nikolay Klopov 2
and Victor Zgoda 1
1Institute of Biomedical Chemistry, Pogodinskaya, 10, 119121 Moscow, Russia
2Petersburg Institute of Nuclear Physics (PNPI) of National Research Center “Kurchatov Institute”,
188300 Gatchina, Russia
3Institute of Biomedical Systems and Biotechnology, Peter the Great St. Petersburg Polytechnic University,
195251 St. Petersburg, Russia
*Correspondence: snaryzhny@mail.ru; Tel.: +7-911-176-4453
Abstract:
The use of tumor markers aids in the early detection of cancer recurrence and prognosis.
There is a hope that they might also be useful in screening tests for the early detection of cancer.
Here, the question of finding ideal tumor markers, which should be sensitive, specific, and reliable,
is an acute issue. Human plasma is one of the most popular samples as it is commonly collected in
the clinic and provides noninvasive, rapid analysis for any type of disease including cancer. Many
efforts have been applied in searching for “ideal” tumor markers, digging very deep into plasma
proteomes. The situation in this area can be improved in two ways—by attempting to find an ideal
single tumor marker or by generating panels of different markers. In both cases, proteomics certainly
plays a major role. There is a line of evidence that the most abundant, so-called “classical plasma
proteins”, may be used to generate a tumor biomarker profile. To be comprehensive these profiles
should have information not only about protein levels but also proteoform distribution for each
protein. Initially, the profile of these proteins in norm should be generated. In our work, we collected
bibliographic information about the connection of cancers with levels of “classical plasma proteins”.
Additionally, we presented the proteoform profiles (2DE patterns) of these proteins in norm generated
by two-dimensional electrophoresis with mass spectrometry and immunodetection. As a next step,
similar profiles representing protein perturbations in plasma produced in the case of different cancers
will be generated. Additionally, based on this information, different test systems can be developed.
Keywords: plasma; biomarker; proteomics; 2DE; proteoform; pattern
1. Introduction
In a broad sense, tumor biomarkers are components that are either produced directly
or indirectly because of a tumor. Moreover, these biomarkers can be common cellular
products that are overproduced by cancer cells or the products of genes that are expressed
only during malignant transformation. Thus, a tumor marker that is present in significant
quantities indicates the presence of cancer. The marker can be present inside the tumor
or enter the bloodstream [
1
,
2
]. This point is fundamentally important, as it allows the
noninvasive examination and treatment of patients with various malignant neoplasms.
The list of biochemical tumor markers known today is large [
2
]. Although some of these
biomarkers have been successfully used in treatment, none of them fully satisfy the so-called
“ideal marker”, which should be highly sensitive, specific, reliable with high predictive
value, and correlate with the stages of tumor development [3].
Therefore, the search for new markers continues. Here, multi-omics technologies
such as genomics, transcriptomics, and metabolomics are very important, but proteomics
plays a central role since tumor biomarkers are mostly proteins. From a proteomic point
of view, the search is based on a comparative analysis of proteomes. These proteomes are
Int. J. Mol. Sci. 2022,23, 11113. https://doi.org/10.3390/ijms231911113 https://www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2022,23, 11113 2 of 35
from body fluids (blood plasma, cerebrospinal fluid, saliva, urine, etc.) or tissues. Here,
human plasma is one of the most popular clinical samples as it provides noninvasive,
rapid analysis for any type of disease. A special human plasma proteome project (HPPP)
project was initiated in 2002 (https://www.hupo.org/plasma-proteome-project accessed on
10 September 2022). Now, this initiative has achieved great success in plasma protein analy-
sis (http://plasmaproteomedatabase.org/index.html accessed on 10 September 2022) [
4
,
5
].
One of the main advantages of using plasma samples is that only a minimally invasive
assay such as a routine blood test analysis is required. To the greatest extent, this certainly
concerns the hematopoietic organs (for instance, the major human plasma proteins are
synthesized mostly in the liver), but also applies to other tissues, and even the brain, which
is separated by the blood–brain barrier. It is expected that the blood plasma proteome
should reflect, to varying degrees, changes in cellular proteomes caused by diseases. In
recent years, biomarker selection guidelines have been developed [
6
–
10
]. Here, the classical
proteomic approaches are used: two-dimensional electrophoresis (2DE), immunodetection,
and mass spectrometry (MS), which have many methodological options that allow highly
productive analysis individually or together in different combinations. Electrophoretic
separation of plasma proteins offers a valuable diagnostic tool, as well as a way to monitor
clinical progress [
11
]. MS measures, with high accuracy, the masses of peptides obtained by
specific hydrolysis of proteins and is very specific. This approach was applied for detecting
ovarian cancer (OC) based on just MS-spectra [
12
]. In addition, MS-based proteomics can
detect and quantify protein variants—proteoforms [
13
]. Ideally, MS-based proteomics can
analyze a whole proteome [
14
–
16
]. A rapid, robust, and reproducible shotgun plasma
proteomics workflow was developed to produce “plasma proteome profiles” [14,17].
Accordingly, there are several directions for proteomics to develop ideal oncomark-
ers. First, we can go deep—find highly specific proteoforms/oncomarkers secreted by
a tumor in low abundancy. Second, go wide—select, and analyze a panel of multiple
proteins/oncomarkers. Third, combine these approaches. There are already some exam-
ples of generation from such panels [
18
]. This strategy can be applied to solid or liquid
biopsies depending on the real situation. Here, the question arises about how to select
these oncomarkers, as the concentration range of putative oncomarkers in plasma is very
wide. The plasma proteome is the most complete version of the whole human proteome.
In addition to the “classical plasma proteins”, it contains tissue proteins plus numerous
individual immunoglobulins [
19
,
20
]. In clinics, a lot of information about the health state is
obtained by analysis of blood proteins. Accordingly, in diagnosis and therapeutic monitor-
ing, human plasma proteome analysis is a promising solution. The major protein, albumin,
accounts for ~50% of the mass of all proteins. Nine proteins (IgG, apolipoprotein A1,
apolipoprotein A2, transferrin, fibrinogen, haptoglobin, alpha1-antitrypsin, transthyretin)
make up 40%, another 12 make up the next 9%, and the rest only 1%. Accordingly, it is
common practice to remove the most abundant proteins (deplete) before deep proteomics
analysis of plasma [21].
Two-dimensional electrophoresis analysis of human plasma proteins has a long history,
where, possibly, the input of L. Anderson and N.G. Anderson is most impressive [
22
–
24
].
There are many publications where the 2DE image of plasma proteins was used as a specific
profile for testing the cancer-related changes in the human body [
25
–
29
]. However, if we
are going to decipher the whole panel of plasma proteins as a combined tumor biomarker,
we need to obtain reliable data about every protein in connection to its response during the
malignancy process. Previously, we started to collect information about the proteoform
profiles of different cellular proteins into a database “2DE pattern” using our original
approaches [
30
]. These approaches are time consuming and labor intensive but allow the
presentation of panoramic data about different proteoforms and could be very useful in
biomarker studies. Here, as a next step in searching for specific oncomarkers, we produced
2DE profiles for the human plasma proteins. The most abundant, “classical plasma proteins”
were selected as they are detected reliably by common proteomics methods.
Int. J. Mol. Sci. 2022,23, 11113 3 of 35
2. Results
In our study, using classical 2DE, sectional 2DE, and semi-virtual 2DE in combination
with liquid chromatography–electrospray ionization tandem mass spectrometry (LC ESI-
MS/MS), we generated 2DE patterns for the most abundant plasma proteins. In Figure 1,
these 2DE images of plasma proteins are presented. The 2DE patterns of more than
100 reliable and confidently detected sets (Supplementary Tables S1 and S2) are presented in
Supplementary Figure S1. We also collected data from the literature about the possibilities of
using these plasma proteins as cancer biomarkers (Table 1) [
20
,
31
]. The detailed information
about these proteins and the 2DE patterns of plasma proteins in norm generated in our
experiments are described below and in the Supplementary File.
Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 4 of 17
reliable and confidently detected sets (Supplementary Tables S1 and S2) are presented in
Supplementary Figure S1. We also collected data from the literature about the possibilities
of using these plasma proteins as cancer biomarkers (Table 1) [20,31]. The detailed
information about these proteins and the 2DE patterns of plasma proteins in norm
generated in our experiments are described below and in the Supplementary File.
(a)
(b)
(c)
Figure 1. Two-dimensional electrophoresis image of depleted plasma proteins taken for the
sectional analysis. (a) A classical annotated 2DE image of plasma proteins; (b) a sectional analysis
of the gel presented in (a). The stained gel was divided into the sections with the predetermined
coordinates, and each section was treated and analyzed by LC ESI-MS/MS (see Materials and
Methods, Section 4.2, 2DE); (c) a semi-virtual 2DE of the major plasma proteins. The plasma proteins
were separated by isoelectrophocusing (IEF), using the 18-cm Immobiline DryStrip 3–11 NL. The
strip was cut to 36 equal sections, and each section was treated and analyzed by LC ESI-MS/MS (see
Materials and Methods, Section 4.2, 2DE). According to the abundance (emPAI) of each protein in
the sections, the graph was plotted. The ball size is proportional to the protein emPAI in each
section.
Table 1. The most abundant plasma proteins related to cancer. Concentration (µg/mL) is presented
according to [16] if otherwise not shown. Abundance (emPAI) was calculated according to data
from the semi-virtual 2DE (Supplementary Table S1). In the column “Cancer”, the references for
cancer-related data are shown (the details are in the Supplementary File).
N.
UniProt
ID (UniProt)
UniProt Name (Gene)
pI/Mw
Leve
µg/mL
µg/mL
EmPAI
Cancer
Figure 1.
Two-dimensional electrophoresis image of depleted plasma proteins taken for the sectional
analysis. (
a
) A classical annotated 2DE image of plasma proteins; (
b
) a sectional analysis of the gel
presented in (
a
). The stained gel was divided into the sections with the predetermined coordinates,
and each section was treated and analyzed by LC ESI-MS/MS (see Materials and Methods, Section 4.2,
2DE); (
c
) a semi-virtual 2DE of the major plasma proteins. The plasma proteins were separated by
isoelectrophocusing (IEF), using the 18-cm Immobiline DryStrip 3–11 NL. The strip was cut to 36 equal
sections, and each section was treated and analyzed by LC ESI-MS/MS (see Materials and Methods,
Section 4.2, 2DE). According to the abundance (emPAI) of each protein in the sections, the graph was
plotted. The ball size is proportional to the protein emPAI in each section.
Int. J. Mol. Sci. 2022,23, 11113 4 of 35
Table 1.
The most abundant plasma proteins related to cancer. Concentration (
µ
g/mL) is presented
according to [
16
] if otherwise not shown. Abundance (emPAI) was calculated according to data
from the semi-virtual 2DE (Supplementary Table S1). In the column “Cancer”, the references for
cancer-related data are shown (the details are in the Supplementary File).
N. UniProt ID (UniProt) UniProt Name (Gene) pI/Mw Leve µg/mL
µg/mL EmPAI Cancer
1
P02763
A1AG1_HUMAN Alpha-1-acid glycoprotein 1 (ORM1) 5.11/21,588 220 116.0 [32–40]
2
P19652
A1AG2_HUMAN Alpha-1-acid glycoprotein 2 (ORM2) 5.12/21,651 220 75.0 [33,41,42]
3
P01009
A1AT_HUMAN Alpha-1-antitrypsin (SERPINA1) 5.37/44,325 350 38.0 [34,43–50]
4
P04217
A1BG_HUMAN Alpha-1B glycoprotein (A1BG) 5.63/51,922 50 44.0 [32,51–53]
5
P01023
A2MG_HUMAN Alpha-2-Macroglobulin (A2M)
5.98/160,810
220 112.0 [27,54–56]
6
P08697
A2AP_HUMAN Alpha-2-antiplasmin
(SERPINF2)5.87/50,451 12 36.0 [57,58]
7
P02750
A2GL_HUMAN Leucine-rich alpha-2-glycoprotein
(LRG1)5.66/34,346 2.7 17.0 [59–61]
8
P01011
AACT_HUMAN Alpha 1-antichymotrypsin
(SERPINA3)5.32/45,266 110 151.0 [62–64]
9
Q15848
ADIPO_HUMAN Adiponectin (ADIPOQ) 5.46/24,544 0.12 2.4 [65]
10
P43652
AFAM_HUMAN Afamin (AFM) 5.58/66,577 320 39.0 [66–70]
11
P02768
ALBU_HUMAN Albumin (ALBU) 5.67/66,472 1600 1207.0 [71–75]
12
P02760
AMBP_HUMAN
Protein AMBP (AMBP)
Alpha-1-microglobulin
Bikunin
5.76/37,115
6.13/20,847
4.89/15,974
48 28.0 [56,76,77]
13
P01019
ANGT_HUMAN Angiotensinogen (AGT) 5.60/49,761 11 46.0 [37]
14
P01008
ANT3_HUMAN Antithrombin-III (SERPINC1) 5.95/49,039 60 155.0 [48,78,79]
15
P02647
APOA1_HUMAN Apolipoprotein A-I (APOAI) 5.27/28,079 310 354.0 [32,80–86]
16
P02652
APOA2_HUMAN Apolipoprotein A-II (APOA2) 5.05/8708 750 285.0 [70,80,87–
91]
17
P06727
APOA4_HUMAN Apolipoprotein A-IV (APOA4) 5.18/43,376 32 78.5 [68,82,92,
93]
18
P04114
APOB_HUMAN Apolipoprotein B-100 (APOB)
6.57/512,858
33 21.0 [52,69,94]
19
P02654
APOC1_HUMAN Apolipoprotein C1 (APOC1) 7.93/6631 77 8.3 [95,96]
20
P02655
APOC2_HUMAN Apolipoprotein C-II (APOC2) 4.58/8204 240 6.1 [20,32]
21
P02656
APOC3_HUMAN Apolipoprotein C-III (APOC3) 4.72/8765 170 6.1 [32,69,91,
95]
22
P05090
APOD_HUMAN Apolipoprotein D (APOD) 5.20/19,303 82 16.8 [32,97]
23
P02649
APOE_HUMAN Apolipoprotein E (APOE) 5.52/34,237 14 52.2 [69,97–101]
24
Q13790
APOF_HUMAN Apolipoprotein F (APOF) 4.40/17,425 4.1 2.0 [102]
25
P02749
APOH_HUMAN Beta-2-glycoprotein 1 (APOH) 8.37/36,255 78 0.1 [103–107]
26
O95445
APOM_HUMAN Apolipoprotein M (APOM) 5.66/21,253 1.5 7.9 [108]
27
P02747
C1QC_HUMAN Complement C1q subcomponent
subunit C (C1QC)8.33/22,813 0.91 1.8 [109]
28
P00736
C1R_HUMAN Complement C1r subcomponent
(C1R)5.76/78,213 4.3 9.7 [110,111]
29
P09871
C1S_HUMAN Complement C1s subcomponent
(C1S)4.85/74,887 5.2 5.6 [112,113]
Int. J. Mol. Sci. 2022,23, 11113 5 of 35
Table 1. Cont.
N. UniProt ID (UniProt) UniProt Name (Gene) pI/Mw Leve µg/mL
µg/mL EmPAI Cancer
30
P05156
CFAI_HUMAN Complement factor I (CFI) 7.38/63,487 0.006 11.9 [49,114]
31
P00751
CFAB_HUMAN Complement factor B (CFB) 6.66/83,001 95 [115] 1.14 [58,69,116,
117]
32
P00746
CFAD_HUMAN Complement factor D (CFD) 6.85/24,405 2.9 7.1 [118]
33
P08603
CFAH_HUMAN Complement factor H (CFH)
6.12/137,053
57 0.24 [32,119–
121]
34
P06681
CO2_HUMAN Complement C2 (C2) 7.57/81,085 35 20.5 [32]
35
P01024
CO3_HUMAN Complement C3 (C3)
6.00/184,951
260 31.1 [58,93,116,
122–125]
36
P0C0L4
CO4A_HUMAN Complement C4-A (C4A)
6.60/190,534
63 [115] 36.3 [52,124,
125]
37
P0C0L5
CO4B_HUMAN Complement C4-B (C4B)
6.83/190,500
90 37.8 [124,125]
38
P01031
CO5_HUMAN Complement C5 (C5)
6.07/186,341
95 35.7 [69,126]
39
P13671
CO6_HUMAN Complement C6 (C6)
6.17/102,412
3.7 15.7 [32,52]
40
P10643
CO7_HUMAN Complement C7 (C7) 6.09/91,115 2.6 17 [69,127]
41
P02748
CO9_HUMAN Complement component C9 (C9) 5.42/60,979 5.2 11.8 [128–130]
42
P00915
CAH1_HUMAN Carbonic anhydrase (CA1) 6.63/28,739 0.59 2.5 [131,132]
43
P08185
CBG_HUMAN Corticosteroid-binding globulin
(SERPINA6)5.64/42,639 1.2 27.9 [133]
44
P15169
CBPN_HUMAN Carboxypeptidase N catalytic chain
(CPN1) 6.88/50,034 0.72 6.4 [134]
45
P08571
CD14_HUMAN Monocyte differentiation antigen
CD14 (CD14) urinary form 5.58/37,215 0.42 4.5 [135,136]
46
P00450
CERU_HUMAN Ceruloplasmin (CP)
5.41/120,085
86 86.7 [34,54,137–
142]
47
P06276
CHLE_HUMAN Cholinesterase (BCHE) 6.33/65,084 0.17 2.97 [143]
48
P10909
CLUS_HUMAN Clusterin (CLU) 5.89/50,063 25 29.9 [32,52,58,
69,144–149]
49
Q96KN2
CNDP1_HUMAN Beta-Ala-His dipeptidase (CNDP1) 5.08/53,864 0.23 2.7 [150–153]
50
P22792
CPN2_HUMAN Carboxypeptidase N subunit 2
(CPN2) 5.54/58,227 2 6.1 [32]
51
P02741
CRP_HUMAN C-reactive protein (CRP)
C-reactive protein (1-205)
5.28/23,047
5.28/22,950 0.26 1.0 [154,155]
52
Q16610
ECM1_HUMAN
Extracellular matrix protein 1 (ECM1)
6.19/58,812 0.77 9.6 [156–158]
53
P23142
FBLN1_HUMAN Fibulin-1 (FBLN1) 5.03/74,291 0.62 11.8 [159–163]
54
O75636
FCN3_HUMAN Ficolin-3 (FCN3) 6.22/30,354 1 11.8 [164–168]
55
P02765
FETUA_HUMAN Alpha-2-HS-glycoprotein (AHSG) 4.53/30,238 82 30.6 [169,170]
56
Q9UGM5
FETUB_HUMAN Fetuin-B (FETUB) 6.52/40,488 0.27 1.8 [171]
57
P02671
FIBA_HUMAN Fibrinogen alpha chain (FGA) 5.79/91,359 0.13 10.9 [32,69,172,
173]
58
P02675
FIBB_HUMAN Fibrinogen beta chain (FGB) 7.95/50,763 130 62.5 [32,173,
174]
59
P02679
FIBG_HUMAN Fibrinogen gamma chain (FGG) 5.24/48,483 98 39.2 [32,69,175–
178]
Int. J. Mol. Sci. 2022,23, 11113 6 of 35
Table 1. Cont.
N. UniProt ID (UniProt) UniProt Name (Gene) pI/Mw Leve µg/mL
µg/mL EmPAI Cancer
60
P02751
FINC_HUMAN Fibronectin (FN1)
5.25/269,259
20 14.1 [48,94,179–
182]
61
P06396
GELS_HUMAN Plasma gelsolin (GSN) 5.72/82,959 16 23.4 [166,183,
184]
62
P22352
GPX3_HUMAN Glutathione peroxidase 3 (GPX3) 7.85/23,464 10 11.7 [185]
63
P69905
HBA_HUMAN Hemoglobin subunit alpha (HBA1) 8.73/15,126 41 1129 [54]
64
P68871
HBB_HUMAN Hemoglobin subunit beta (HBB) 6.81/15,867 30 847.0 [54,186]
65
P02790
HEMO_HUMAN Hemopexin (HPX) 6.43/49,295 180 165.0 [177,187–
189]
66
P05546
HEP2_HUMAN Heparin Cofactor 2 (SERPIND1) 6.26/54,960 4.3 43.0 [58,189–
192]
67
P00738
HPT_HUMAN
Haptoglobin (Zonulin) (HP)
haptoglobin alpha 1 chain
haptoglobin alpha 2 chain
haptoglobin beta chain
6.13/43,349
5.23/93,55
5.57/15,946
6.32/27,265
210 323.0 [166,193–
199]
68
P00739
HPTR_HUMAN Haptoglobin-related protein (HPR) 6.63/39,030 41 [200] 105.0 [201]
69
P04196
HRG_HUMAN Histidine-rich glycoprotein (HRG) 7.03/57,660 35 24.0 [202,203]
70
P05155
IC1_HUMAN Plasma protease C1 inhibitor
(SERPING1)5.97/52,843 12 9.4 [204,205]
71
P19827
ITIH1_HUMAN Inter-alpha-trypsin inhibitor heavy
chain H1 (ITIH1)6.33/71,415 24 25.0 [29,206–
210]
72
Q06033
ITIH3_HUMAN Inter-alpha-trypsin inhibitor heavy
chain H3 (ITIH3)5.01/69,360 2 7.7 [207]
73
Q14624
ITIH4_HUMAN Inter-alpha-trypsin inhibitor heavy
chain H4 (ITIH4)5.92/70,586 42 41.6 [29,207]
74
P29622
KAIN_HUMAN Kallistatin (SERPINA4) 7.88/46,355 1.1 81.8 [211]
75
P01042
KNG1_HUMAN Kininogen 1 (KNG1) 6.23/69,897 28 7.7 [212–214]
76
P04180
LCAT_HUMAN Phosphatidylcholine-sterol
acyltransferase (LCAT)5.71/47,084 0.22 1.8 [171]
77
P51884
LUM_HUMAN Lumican (LUM) 6.17/36,661 4 6.4 [166,215–
217]
78
P11226
MBL2_HUMAN Mannose-binding protein C (MBL2) 5.40/24,021 0.07 6.4 [171,218,
219]
79
P36955
PEDF_HUMAN Pigment epithelium-derived factor
(SERPINF1)5.90/44,388 7.2 14.5 [220]
80
Q96PD5
PGRP2_HUMAN N-acetylmuramoyl-L-alanine
amidase (PGLYRP2)7.64/59,980 14 4.0 [171,221,
222]
81
P80108
PHLD_HUMAN Phosphatidylinositol-glycan-specific
phospholipase D (GPLD1)5.78/89,811 4 3.7 [32,223,
224]
82
P00747
PLMN_HUMAN
Plasminogen (PLG)
Plasmin heavy chain A
Angiostatin
Plasmin heavy chain A, short form
Plasmin light chain
7.08/88,432
6.79/63,245
8.30/44,053
7.44/54,341
7.67/25,205
25 81.0 [225,226]
83
P27169
PON1_HUMAN Serum paraoxonase/arylesterase 1
(PON1)5.08/39,600 7.7 43.4 [79,227–
232]
Int. J. Mol. Sci. 2022,23, 11113 7 of 35
Table 1. Cont.
N. UniProt ID (UniProt) UniProt Name (Gene) pI/Mw Leve µg/mL
µg/mL EmPAI Cancer
84
P27918
PROP_HUMAN Properdin (CFP) 8.33/48,494 0.33 1.7 [233]
85
P07225
PROS_HUMAN Vitamin K-dependent protein S
(PROS1)5.17/70,645 1.7 7.7 [234]
86
P02753
RET4_HUMAN Plasma retinol-binding protein 4
(PRBP)5.27/21,072 580 39.3 [235–237]
87
P0DJI8
SAA1_HUMAN Serum amyloid A-1 (SAA1) 5.89/11,683 7.4 4.3 [32,34,238–
240]
88
P02743
SAMP_HUMAN
Serum amyloid P-component (APCS)
6.12/23,259 8.7 39.5 [241]
89
P04278
SHBG_HUMAN Sex hormone-binding globulin
(SHBG)5.83/40,468 0.26 5.8 [242,243]
90
P05109
S10A8_HUMAN Protein S100-A8 (S100A8) 6.50/10,835 0.27 0.9 [244–246]
91
P06702
S10A9_HUMAN Protein S100-A9 (S100A9) 5.71/13,242 1.9 2.6 [244,247]
93
P05452
TETN_HUMAN Tetranectin (CLEC3B) 5.80/20,139 58 31.5 [32,248,
249]
94
P05543
THBG_HUMAN Thyroxine-binding globulin
(SERPINA7) 5.76/44,102 1.3 12.5 [250,251]
95
P00734
THRB_HUMAN Prothrombin (F2) 5.23/65,308 27 24.8 [252,253]
96
P02787
TRFE_HUMAN Serotransferrin (TF) 6.70/75,195 360 41.8 [25,254]
97
P02766
TTHY_HUMAN Transthyretin (TTR) 5.31/13,761 770 23.9 [32,95,177,
255–257]
98
P02774
VTDB_HUMAN Vitamin D-binding protein (GC) 5.16/51,197 57 181.4 [32,258,
259]
99
P04004
VTNC_HUMAN Vitronectin (VTN) 5.47/52,278 35 22.7 [25,32,52,
260]
100 P25311
ZA2G_HUMAN Zinc-alpha2-glycoprotein (AZGP1) 5.58/32,145 31 31.5 [261–264]
2.1. ALPHA-1-ACID GLYCOPROTEIN 1 (A1AG1_HUMAN)
The two-dimensional electrophoresis pattern of AGP-1 represents a chain of spots
in the pI-range from 3 to 5 (Supplementary Figure S1). This pattern is well-represented
in the SWISS-2DPAGE (pI/Mw: 4.11–4.29/43–46,000) [
22
]. Such a pattern is a result of
heavy glycosylation (82 N-linked glycans at 6 sites), phosphorylation (2 sites), acetylation
(2 sites), ubiquitylation (1 site) (https://www.uniprot.org/uniprot/P02763 accessed on
10 September 2022).
2.2. ALPHA-1-ACID GLYCOPROTEIN 2 (A1AG2_HUMAN)
The two-dimensional electrophoresis pattern of AGP-2 is very similar to AGP-1
(Supplementary Figure S1) and in SWISS-2DPAGE is overlapped with AGP-1 pattern [
22
].
AGP-2 can be glycosylated (99 N-linked glycans at 7 sites) and acetylated (1 site). (https:
//www.uniprot.org/uniprot/P19652 accessed on 10 September 2022).
2.3. ALPHA-1-ANTITRYPSIN (A1AT_HUMAN)
The two-dimensional electrophoresis pattern of Serpin A1 represents a chain of spots
in the pI-range 4.5–5.1 (Supplementary Figure S1) [
265
] that is a result of multiple N-linked
glycosylations (112 N-linked glycans at 5 sites, 5 O-linked glycans at 6 sites), phosphory-
lation (13 sites), and acetylation (17 sites) (https://www.phosphosite.org/ accessed on
10 September 2022) [
266
]. Accordingly, in SWISS-2DPAGE, 22 spots of serpin A1 are present
(pI/Mw: 4.87–5.10/48–108,000) [22].
Int. J. Mol. Sci. 2022,23, 11113 8 of 35
2.4. ALPHA-1B GLYCOPROTEIN (A1BG_HUMAN)
The two-dimensional electrophoresis pattern of alpha-1-B glycoprotein represents a
long chain of spots in the pI-range 4–6 and Mw ~ 54,000 (Supplementary Figure S1) that
is a result of heavy glycosylation (24 N-linked glycans at 4 sites, 2 O-linked glycans at
1 site) and phosphorylation (1 site) (https://glyconnect.expasy.org/browser/proteins/780
accessed on 10 September 2022). In SWISS-2DPAGE, alpha-1-B glycoprotein is represented
as a chain of six spots (pI/Mw: 4.99–5.25/73–76,000) [22].
2.5. ALPHA-2-MACROGLOBULIN (A2MG_HUMAN)
The two-dimensional electrophoresis pattern of alpha-2-M represents a chain of heavy
Mw spots (mostly in the pI-range 5.8–6.3) (Supplementary Figure S1). This pattern is also
well-represented in the SWISS-2DPAGE https://world-2dpage.expasy.org/ accessed on
10 September 2022 [
22
]. Alpha-2-M has eight sites of O-GalNAc and eight sites of N-GlcNAc
(https://glygen.org/protein/P01023#glycosylation accessed on 10 September 2022).
2.6. ALPHA-2-ANTIPLASMIN (A2AP_HUMAN)
The two-dimensional electrophoresis pattern of
α
2AP represents a chain of spots in the
pI-range 4–6 with Mw ~ 50,000 (Supplementary Figure S1) that is a result of glycosylation
(4 N-linked glycans at 1 site, 3 O-linked glycans at 4 sites), phosphorylation (7 sites), and
ubiquitylation (2 sites) (https://glygen.org/protein/P08697#glycosylation accessed on
10 September 2022). In SWISS-2DPAGE, alpha-2-antiplasmin is represented as a chain of
seven spots (pI/Mw: 4.87–5.17/66–74,000).
2.7. LEUCINE-RICH ALPHA-2-GLYCOPROTEIN (A2GL_HUMAN)
The two-dimensional electrophoresis pattern of LRG1 represents a chain of spots in
the pI-range 3.5–5.0 with Mw ~ 40,000–50,000 (Supplementary Figure S1). This pattern
is well-represented in the SWISS-2DPAGE https://world-2dpage.expasy.org/ accessed
on 10 September 2022 and has a characteristic for multiple glycosylation profiles, where
acidic spots have higher Mw [
22
]. LRG1 has at least six sites of glycosylation: one is
O-GalNAc and five are N-GlcNAc (https://www.uniprot.org/uniprot/P02750 accessed on
10 September 2022).
2.8. ALPHA-1-ANTICHYMOTRYPSIN (AACT_HUMAN)
The two-dimensional electrophoresis pattern of ACT represents a chain of spots
in the pI-range 4.0–5.0 and Mw 50–60,000 (Supplementary Figure S1). This pattern is
also well-represented in the SWISS-2DPAGE, where two chains (20 spots) of both ACT
forms are presented [
22
]. ACT has seven sites of N-GlcNAc and four sites of O-GalNAc
(https://glygen.org/protein/P01011#glycosylation accessed on 10 September 2022).
2.9. ADIPONECTIN (ADIPO_HUMAN)
The two-dimensional electrophoresis pattern of adiponectin represents a chain of spots
in the pI-range 5.0–5.5 and Mw 26,000 (Supplementary Figure S1). There are six sites of
O-linked glycosylation and two sites of phosphorylation in adiponectin (https://glygen.
org/protein/Q15848#glycosylation accessed on 10 September 2022).
2.10. AFAMIN (AFAM_HUMAN)
The two-dimensional electrophoresis pattern of adiponectin represents a chain of spots
in the pI-range 4.5–6.0 and Mw ~ 70,000 (Supplementary Figure S1). There are six sites
of N-linked glycosylation in afamin, and more than 90% of the glycans are sialylated
(https://glygen.org/protein/P43652#glycosylation accessed on 10 September 2022).
2.11. ALBUMIN (ALBU_HUMAN)
The two-dimensional electrophoresis pattern of albumin represents a chain of spots
in the pI-range 5.5–6.5 and Mw ~ 70,000 (Supplementary Figure S1). Its pattern is also
Int. J. Mol. Sci. 2022,23, 11113 9 of 35
well-represented in the SWISS-2DPAGE [
22
]. Albumin can be modified by N-linked glycans
at one site, 7 O-linked glycans at 11 sites, phosphorylated at multiple sites (at least 15),
and acetylated (1 site) (https://glygen.org/protein/P02768#glycosylation accessed on
10 September 2022).
2.12. PROTEIN AMBP (AMBP_HUMAN)
The two-dimensional electrophoresis pattern of AMBP represents two groups of spots
in the pI-range 4.0–6.5: alpha-1-microglobulin with Mw ~ 26,000 and inter-alpha-trypsin
inhibitor light chain/bikunin that is assembled in a high Mw complex by a chondroitin-like
glycosaminoglycan (GAG) cross-linking with Mw 120,000 (Supplementary Figure S1). In
the SWISS-2DPAGE, AMBP is represented only by the chain of three alpha-1-microglobulin
spots. AMBP can be glycosylated (31 N-Linked glycans at 2 sites, 7 O-Linked glycans at
3 sites), phosphorylated (3 sites), and acetylated (1 site) (https://www.phosphosite.org/
accessed on 10 September 2022).
2.13. ANGIOTENSINOGEN (ANGT_HUMAN)
The two-dimensional electrophoresis pattern of angiotensinogen represents a chain
of spots in the pI-range 4.0–6.4 and Mw ~ 50,000 (Supplementary Figure S1). In the
SWISS-2DPAGE, angiotensinogen is represented by one spot (pI/Mw: 5.07/58,973). It
was reported that there were 20 N-linked glycans at 3 sites and 1 O-linked glycan (1 site)
(https://glygen.org/protein/P01019#glycosylation accessed on 10 September 2022).
2.14. ANTITHROMBIN-III (ANT3_HUMAN)
The two-dimensional electrophoresis pattern of ATIII represents a chain of eight
spots in the pI-range 4.5–6.0 and Mw ~ 50,000 (Supplementary Figure S1). In the SWISS-
2DPAGE, only two spots are presented (pI/Mw: 5.20/58,973 and 5.27/58,653) for ATIII [
22
].
The protein can be glycosylated (24 N-linked glycans at 4 sites, 2 O-linked glycans at
1 site), phosphorylated (9 sites), and ubiquitinated (1 site) (https://www.phosphosite.org/
accessed on 10 September 2022).
2.15. APOLIPOPROTEIN A-I (APOA1_HUMAN)
The two-dimensional electrophoresis pattern of apoA-I represents a chain of spots in
the pI-range 4.5–6.5 and Mw ~ 26,000 (Supplementary Figure S1). In the SWISS-2DPAGE,
nine spots are presented (chain of five spots pI/Mw: 4.99–5.48/~23,000 and four spots with
Mw ~ 8000–9000) [
22
]. The protein can be heavily phosphorylated (13 sites), acetylated
(13 sites), ubiquitinated (7 sites), succinylated (3 sites), or glycosylated (2 sites) (https:
//www.phosphosite.org/ accessed on 10 September 2022).
2.16. APOLIPOPROTEIN A-II (APOA2_HUMAN)
The two-dimensional electrophoresis pattern of apoA-II represents a chain of spots in
the pI-range 4.5–6.0 and Mw ~ 9000 (Supplementary Figure S1). In the SWISS-2DPAGE,
two spots are presented (pI/Mw: 4.74/12,520 and 4.71/7250) [
22
]. ApoA-II can be gly-
cosylated (3 O-linked glycans at 3 sites), phosphorylated (7 sites), acetylated (1 site), or
succinylated (1 site).
2.17. APOLIPOPROTEIN A-IV (APOA4_HUMAN)
The two-dimensional electrophoresis pattern of apoA-IV represents a chain of spots in
the pI-range 4.5–6.0 and Mw ~ 40,000 (Supplementary Figure S1). In the SWISS-2DPAGE,
six spots are presented (pI/Mw: 5.05–5.10/~43,000 (3 spots), 5.11/21,945, and 4.87–4.97/9–
10,000 (3 spots)). ApoA-IV can be glycosylated (1 O-linked glycan at 1 site), phosphorylated
(7 sites), acetylated (9 site), or ubiquitinated (1 site) (https://www.phosphosite.org/ ac-
cessed on 10 September 2022).
Int. J. Mol. Sci. 2022,23, 11113 10 of 35
2.18. APOLIPOPROTEIN B-100 (APOB_HUMAN)
The two-dimensional electrophoresis pattern of apo B-100 represents a long chain of
spots in the pI-range 3.5–7.5 and heavy Mw > 120,000 (Supplementary Figure S1). Apo
B-100 can be glycosylated (28 sites, 82 N-linked glycans at 16 sites, 1 O-linked glycan at
7 sites), phosphorylated (43 sites), acetylated (4 sites), or ubiquitinated (8 sites) (https:
//www.phosphosite.org/ accessed on 10 September 2022).
2.19. APOLIPOPROTEIN C-I (APOC1_HUMAN)
The two-dimensional electrophoresis pattern of apo-CI represents a chain of spots
in the pI-range 7.8–8.5 and Mw ~ 6000 (Supplementary Figure S1). This protein can be
acetylated (3 sites) and ubiquitinated (4 sites) (https://www.phosphosite.org/ accessed on
10 September 2022).
2.20. APOLIPOPROTEIN C-II (APOC2_HUMAN)
The two-dimensional electrophoresis pattern of apo-CII represents a chain of spots in
the pI-range 4.8–5.2 and Mw ~ 8000 (Supplementary Figure S1). In the SWISS-2DPAGE,
two spots for apo-CII are presented (pI/Mw: 4.51/9976 and 4.58/9248). This protein can
be acetylated (5 sites), ubiquitinated (2 sites) (https://www.phosphosite.org/ accessed on
10 September 2022), and glycosylated (3 O-linked glycans at 4 sites) (https://www.glygen.
org/protein/P02655 accessed on 10 September 2022).
2.21. APOLIPOPROTEIN C-III (APOC3_HUMAN)
The two-dimensional electrophoresis pattern of apoC-III represents a chain of spots in
the pI-range 3.8–6.1 and Mw ~ 9000 (Supplementary Figure S1). In the SWISS-2DPAGE, only
one spot for apoC-III is presented (pI/Mw: 4.63/8528). This protein can be phosphorylated
(7 sites) acetylated (1 site), ubiquitinated (1 site) (https://www.phosphosite.org/ accessed
on 10 September 2022), and glycosylated (4 O-linked glycans at 1 site) (https://www.
glygen.org/protein/P02655 accessed on 10 September 2022).
2.22. APOLIPOPROTEIN D (APOD_HUMAN)
The two-dimensional electrophoresis pattern of apoD represents an unusual set of
spots in the pI-range 3.5–6.5 and Mw from ~15,000 to ~26,000 and 80,000 (Supplementary
Figure S1). In the SWISS-2DPAGE, a cluster of 12 spots for apoD is presented (pI/Mw: 4.44–
4.78/27–32,000). ApoD can be heavily glycosylated (115 N-linked glycans at 2 sites, 1 O-
linked glycan at 1 site) and phosphorylated (1 site) (https://www.uniprot.org/uniprotkb/
P05090/entry accessed on 10 September 2022).
2.23. APOLIPOPROTEIN E (APOE_HUMAN)
The two-dimensional electrophoresis pattern of apoE represents a chain of spots in
the pI-range 4.5–6.5 and Mw ~ 35,000 (Supplementary Figure S1). In the SWISS-2DPAGE,
there is a chain of three spots (pI/Mw: 5.24–5.49/34–35,320). ApoE can be glycosylated
(6 O-linked glycans at 6 sites), phosphorylated (9 sites), acetylated (1 site), and ubiquitinated
(5 sites) (https://www.phosphosite.org/ accessed on 10 September 2022).
2.24. APOLIPOPROTEIN F (APOF_HUMAN)
The two-dimensional electrophoresis pattern of apo-F represents a set of spots in the
pI-range 3.5–4.2 and Mw from ~15,000 to ~32,000 (Supplementary Figure S1). ApoF can
be glycosylated (16 N-linked glycans at 3 sites, 6 O-linked glycans at 5 sites), phospho-
rylated (1 site), and ubiquitinated (2 sites) (https://www.phosphosite.org/ accessed on
10 September 2022).
2.25. BETA-2-GLYCOPROTEIN 1 (APOH_HUMAN)
The two-dimensional electrophoresis pattern of apo-H represents a chain of spots in
the pI-range 6.2–8.4 and Mw ~ 52,000 (Supplementary Figure S1), which is much higher
Int. J. Mol. Sci. 2022,23, 11113 11 of 35
than the theoretical one because of heavy glycosylation (85 N-linked annotations at 4 sites
and 3 O-linked annotations at 3 sites) (https://glygen.org/protein/P02749#glycosylation
accessed on 10 September 2022).
2.26. APOLIPOPROTEIN M (APOM_HUMAN)
The two-dimensional electrophoresis pattern of apoM represents a chain of spots in
the pI-range 4.5–6.5 and Mw ~ 22,000 (Supplementary Figure S1). There are 13 N-linked
annotations at 1 site (N135) in apoM (https://glygen.org/protein/O95445#glycosylation
accessed on 10 September 2022).
2.27. Complement System
The results of several studies suggest that changes in the complement system can not
only promote an antitumor response but can also influence tumor development through
proliferation, survival, angiogenesis, and invasiveness [
267
,
268
]. The presence of many
complement components with different functions makes the study of this system very
difficult [
269
]. In any case, it is becoming clear that complement activation stimulates
carcinogenesis and protects against immune destruction, although it has long been believed
that the complement system helps the body identify and eliminate transformed cells.
Moreover, the complement is activated by different mechanisms in the case of different
types of cancer, and the results of activation may be different for different types of cancer
or over time for the same tumor [270–272].
2.27.1. C1R (C1R_HUMAN)
The two-dimensional electrophoresis pattern represents only a chain of spots of the
complement C1r subcomponent in the pI-range 4.5–6.2 and Mw ~ 80,000 (Supplementary
Figure S1) that corresponds to only a complement C1r subcomponent. The cleaved heavy
and light chains were not detected. There are 25 N-linked glycosylation annotations at
four sites and one phosphorylation site in the complement C1r subcomponent (https:
//glygen.org/protein/P00736#glycosylation accessed on 10 September 2022).
2.27.2. C1S (C1S_HUMAN)
The two-dimensional electrophoresis-pattern represents a chain of five spots of C1s
in the pI-range from 4.0 to 4.9 and Mw ~ 80,000 (Supplementary Figure S1). The cleaved
heavy and light chains were not detected. There are seven N-linked glycans at two sites
(https://glygen.org/protein/P09871#glycosylation accessed on 10 September 2022).
2.27.3. COMPLEMENT C1qC (C1QC_HUMAN)
The two-dimensional electrophoresis pattern of C1q represents a long horizontal chain
of spots in the pI-range 3.0–9.5 with Mw ~ 23,000 and a vertical chain of heavy complexes
(Mw 23,000 and up) with pI ~ 9.0 (Supplementary Figure S1). It was reported there was
only one O-linked glycosylation of C1q (https://glygen.org/protein/P02747#glycosylation
accessed on 10 September 2022).
2.27.4. COMPLEMENT FACTOR I (CFAI_HUMAN)
The two-dimensional electrophoresis pattern of the complement factor I represents
the chains of many spots in the pI-range 4.5–6.8 from Mw ~ 64,000 (complement factor I) to
Mw ~ 30,000 (the complement factor I heavy and light chains) (Supplementary Figure S1).
In the SWISS-2DPAGE, the complement factor I is represented only by one spot (pI/Mw:
5.03/37,900). There are 57 N-linked glycosylation annotations at 6 sites for the complement
factor I (https://glygen.org/protein/P05156#glycosylation accessed on 10 September 2022).
2.27.5. COMPLEMENT FACTOR B (CFAB_HUMAN)
The two-dimensional electrophoresis pattern of the complement factor B represents
the chains of spots in the pI-range 4.5–6.8 with Mw ~ 90,000 (Supplementary Figure S1). The
Int. J. Mol. Sci. 2022,23, 11113 12 of 35
cleaved heavy and light chains were not detected. In the SWISS-2DPAGE, the complement
factor B is represented by a chain of six spots (pI 5.88–6.28, Mw ~ 100,000). There are
19 N-linked glycans (4 sites), and 3 O-linked glycans (3 sites) in the complement factor B
(https://glygen.org/protein/P00751#glycosylation accessed on 10 September 2022).
2.27.6. COMPLEMENT FACTOR D (CFAD_HUMAN)
The two-dimensional electrophoresis pattern of the complement factor D represents
two spots (pI ~8.0, Mw 25,000) (Supplementary Figure S1). The protein can be phos-
phorylated (2 sites), glycosylated (2 sites), ubiquitinated (2 sites), and methylated (1 site)
(https://www.phosphosite.org accessed on 10 September 2022).
2.27.7. COMPLEMENT FACTOR H (CFAH_HUMAN)
The two-dimensional electrophoresis pattern of the complement factor D represents a
long chain of spots in the pI-range 5.5–7 with Mw ~ 140,000 (Supplementary Figure S1).
It was reported there were 62 N-linked glycans in 9 sites in the complement factor D
(https://glygen.org/protein/P08603#glycosylation accessed on 10 September 2022).
2.27.8. COMPLEMENT C2 (CO2_HUMAN)
The two-dimensional electrophoresis pattern of the complement C2 represents a
chain of spots in the pI-range 6–7 with Mw ~ 80,000 (Supplementary Figure S1). It was
reported there were 33 N-linked glycosylations at 9 sites and one phosphorylation (S266)
of the complement C2 (https://glygen.org/protein/P06681#glycosylation accessed on
10 September 2022).
2.27.9. COMPLEMENT C3 (CO3_HUMAN)
The two-dimensional electrophoresis pattern of the complement C3 represents acluster
of spots in the pI-range 3.5–7.5 and Mw from ~30,000 to 180,000 (Supplementary Figure S1).
In the SWISS-2DPAGE, there are the complement C3 beta chain (5 spots with pI 6.81–6.98,
Mw ~ 71,000) and the complement C3dg fragment (a spot with pI 4.84 and Mw 40,915).
There are 50 N-linked glycans at 4 sites, 2 O-linked glycans at 2 sites, and 12 phosphorylation
sites (https://glygen.org/protein/P01024#glycosylation accessed on 10 September 2022).
2.27.10. COMPLEMENT C4-A (CO4A_HUMAN)
The two-dimensional electrophoresis pattern of the complement C4-A represents a wide
cluster of spots in the pI-range 3.0–10.0 and Mw from ~25,000 to 190,000 (Supplementary Figure S1).
There are 35 N-linked glycans (4 sites), 6 O-linked glycans (4 sites), and 3 phosphoserine sites
(https://glygen.org/protein/P0C0L4#glycosylation accessed on 10 September 2022).
2.27.11. COMPLEMENT C4-B (C4B) (CO4B_HUMAN)
The two-dimensional electrophoresis pattern of the complement C4-B represents a
wide cluster of spots in the pI-range 3.0–10.0 and Mw from ~35,000 to 19,0000
(Supplementary Figure S1). In the SWISS-2DPAGE, only a complement C4 gamma chain
(2 spots with pI/Mw: 6.41/31,942 and 6.54/31,735) was detected. It was reported there
were 34 N-linked glycans (4 sites) and 1 O-linked glycan (1 site) in the complement C4-B
(https://glygen.org/protein/P0C0L5#glycosylation accessed on 10 September 2022).
2.27.12. COMPLEMENT C5 (CO5_HUMAN)
The two-dimensional electrophoresis pattern of the complement C4-B represents
a wide cluster of spots in the pI-range 5.0–6.8 and Mw ~ 70,000–190,000 (Supplemen-
tary Figure S1). It was reported there were eight N-linked glycans at three sites (https:
//glygen.org/protein/P01031#glycosylation accessed on 10 September 2022).
Int. J. Mol. Sci. 2022,23, 11113 13 of 35
2.27.13. COMPLEMENT C6 (CO6_HUMAN)
The two-dimensional electrophoresis pattern of the complement C6 represents a chain
of spots in the pI-range 4.0–6.5 and Mw ~ 100,000 (Supplementary Figure S1). It was
reported there were 6 C-linked annotations at 6 sites, 12 N-linked annotations at 4 sites, 2 O-
linked annotations at 2 sites (https://glygen.org/protein/P13671#glycosylation accessed
on 10 September 2022), and 5 sites of phosphorylation (https://www.phosphosite.org/
accessed on 10 September 2022).
2.27.14. COMPLEMENT C7 (CO7_HUMAN)
The two-dimensional electrophoresis pattern of the complement C7 represents a chain
of spots in the pI-range 4.5–6.5 and Mw ~ 100,000 (Supplementary Figure S1). It was
reported there were three N-linked glycans at two sites and two O-linked glycans at one
site (https://glygen.org/protein/P10643#glycosylation accessed on 10 September 2022).
2.27.15. COMPLEMENT C9 (CO9_HUMAN)
The two-dimensional electrophoresis pattern of the complement C9 represents a chain
of spots in the pI-range 4.5–5.5 and Mw ~ 60,000 (Supplementary Figure S1). The protein can
be glycosylated (10 N-linked glycans at 2 sites, 4 O-linked glycans at 5 sites), phosphorylated
(10 sites), acetylated (1 site), and ubiquitinated (3 sites) (https://www.phosphosite.org/
accessed on 10 September 2022).
2.28. CARBONIC ANHYDRASE I (CAH1_HUMAN)
The two-dimensional electrophoresis pattern of CAB represents a chain of spots in
the pI-range 5–7 and Mw ~ 28,000 (Supplementary Figure S1). It was reported there was
glycosylation (2 O-linked at 2 sites) (https://glygen.org/protein/P00915#glycosylation
accessed on 10 September 2022), phosphorylation (11 sites), acetylation (5 sites), and
ubiquitylation (2 sites) (https://www.phosphosite.org/ accessed on 10 September 2022).
2.29. CORTICOSTEROID-BINDING GLOBULIN (CBG_HUMAN)
The two-dimensional electrophoresis pattern of CBG represents a chain of spots
in the pI-range 3.7–5.1 and Mw ~ 50,000 (Supplementary Figure S1). It was reported
there were 48 N-linked glycosylations at 6 sites and 2 O-linked glycosylations at 1 site
(https://glygen.org/protein/P08185#glycosylation accessed on 10 September 2022).
2.30. CARBOXYPEPTIDASE N CATALYTIC CHAIN (CBPN_HUMAN)
The two-dimensional electrophoresis pattern of CBPN represents a chain of spots in
the pI-range 4.5–7 and Mw ~ 50,000 (Supplementary Figure S1). It was reported there were
32 N-linked glycans at 5 sites and 2 O-linked glycans at 1 site https://glygen.org/protein/
P08185#glycosylation accessed on 10 September 2022.
2.31. MONOCYTE DIFFERENTIATION ANTIGEN CD14 (CD14_HUMAN)
The two-dimensional electrophoresis pattern of CD14 represents a chain of spots in
the pI-range 4.5–5.8 and Mw ~ 40,000 (Supplementary Figure S1). It was reported there
were 26 N-linked glycans at 2 sites and 4 O-linked glycans at 3 sites (https://glygen.org/
protein/P08571#glycosylation accessed on 10 September 2022).
2.32. CERULOPLASMIN (CERU_HUMAN)
The two-dimensional electrophoresis pattern of ceruloplasmin represents a chain of
spots in the pI-range 4.0–6.2 and Mw ~ 120,000 (Supplementary Figure S1). In the SWISS-
2DPAGE, 3 chains of 27 spots with pI 4.96–5.24 and Mw ~ 120–16,0000 are present. There
are 237 N-linked annotations at 8 sites, 10 O-linked annotations at 7 sites of glycosylation,
and 3 sites of phosphorylation for ceruloplasmin (https://glygen.org/protein/P00450
#glycosylation accessed on 10 September 2022).
Int. J. Mol. Sci. 2022,23, 11113 14 of 35
2.33. CHOLINESTERASE (CHLE_HUMAN)
The two-dimensional electrophoresis pattern of cholinesterase represents a chain of
five spots in the pI-range 4.5–5.2 and Mw ~ 65,000 (Supplementary Figure S1). There are
34 N-linked annotations at 12 sites, one O-linked annotation for glycosylation, and phos-
phorylation at S226 for cholinesterase (https://glygen.org/protein/P06276#glycosylation
accessed on 10 September 2022).
2.34. CLUSTERIN (CLUS_HUMAN)
The two-dimensional electrophoresis pattern of ceruloplasmin represents a chain
of 18 spots in the pI-range 4.5–6.5 and Mw ~ 35,000 (Supplementary Figure S1). In the
SWISS-2DPAGE, 17 spots with pI 4.73–5.07 and Mw ~ 35–39,000 are shown. Clusterin
is heavily glycosylated (149 N-linked glycans at 6 sites, O-linked glycan at 1 site) and
phosphorylated (4 sites) (https://glygen.org/protein/P10909#glycosylation accessed on
10 September 2022).
2.35. BETA-ALA-HIS DIPEPTIDASE (CNDP1_HUMAN)
The two-dimensional electrophoresis pattern of beta-Ala-His dipeptidase represents
two spots around pI 5.0 and Mw ~ 54,000 (Supplementary Figure S1). It was reported there
were 18 N-linked glycans at 1 site, 1 O-linked glycan at 2 sites, and phosphorylation at S219
(https://glygen.org/protein/Q96KN2#glycosylation accessed on 10 September 2022).
2.36. CARBOXYPEPTIDASE N SUBUNIT 2 (CPN2_HUMAN)
The two-dimensional electrophoresis pattern of carboxypeptidase N subunit 2 rep-
resents nine spots with pI 3.5–5.5 and Mw ~ 65,000 (Supplementary Figure S1). It is
known there were 10 N-linked glycans at 3 sites in carboxypeptidase N subunit 2 (https:
//www.glygen.org/protein/P22792 accessed on 10 September 2022).
2.37. C-REACTIVE PROTEIN (CRP_HUMAN)
The two-dimensional electrophoresis pattern of CRP represents a single spot (pI/Mw:
5.2/24,000) (Supplementary Figure S1). In the SWISS-2DPAGE, a similar situation exists—a
single spot (pI/Mw: 5.12/23,760). Thus far, it was reported there was only one PTM
(a pyroglutamic acid, Q19) for CRP (https://www.uniprot.org/uniprotkb/P02741/entry
accessed on 10 September 2022).
2.38. EXTRACELLULAR MATRIX PROTEIN I (ECM1_HUMAN)
The two-dimensional electrophoresis pattern of ECM1 represents a spot (pI/Mw:
6.0/60,000) (Supplementary Figure S1). It was reported there were 21 N-linked glycans
at 4 sites, and 3 O-linked glycans at 6 sites (https://www.glygen.org/protein/Q16610
accessed on 10 September 2022).
2.39. FIBULIN-1 (FBLN1_HUMAN)
The two-dimensional electrophoresis pattern of FIBL-1 represents a chain of four
spots (pI/Mw: 4.5–5.2/75,000) (Supplementary Figure S1). It was reported there were
10 N-linked glycans at two sites, one O-linked glycan, and one phosphorylation at S147
(https://www.glygen.org/protein/P23142 accessed on 10 September 2022).
2.40. FICOLIN-3 (FCN3_HUMAN)
The two-dimensional electrophoresis-pattern of ficolin-3 represents a chain of five spots
(pI/Mw: 5.8–6.5/30,000) (Supplementary Figure S1). It was reported there were five N-
linked annotation(s) at one site (https://www.glygen.org/protein/O75636 accessed on
10 September 2022).
Int. J. Mol. Sci. 2022,23, 11113 15 of 35
2.41. ALPHA-2-HS-GLYCOPROTEIN (FETUA_HUMAN)
The two-dimensional electrophoresis-pattern of fetuin-A represents a set of proteo-
forms (pI/Mw: 3.7–6.3/~40,000-up) (Supplementary Figure S1). In the SWISS-2DPAGE,
15 spots (pI/Mw: 4.56–4.77/52–58,000) are shown. The protein is heavily glycosylated (126
N-linked annotations at 2 sites, 43 O-linked annotations at 14 sites) and phosphorylated
(https://www.glygen.org/protein/P02765 accessed on 10 September 2022).
2.42. FETUIN-B (FETUB_HUMAN)
The two-dimensional electrophoresis pattern of fetuin-B represents a chain of proteoforms
(pI/Mw: 5.0–6.3/50,000-up) (Supplementary Figure S1). The protein is heavily glycosylated
(26 N-linked annotations at 3 sites, 8 O-linked annotation(s) at 6 sites) and phosphorylated
(https://www.glygen.org/protein/Q9UGM5 accessed on 10 September 2022).
2.43. FIBRINOGEN ALPHA CHAIN (FIBA_HUMAN)
The two-dimensional electrophoresis pattern of FBA represents several sets of chains
with pI 5.0–7.5 (Mw ~ 30–35,000, Mw ~ 64–83,000, Mw ~ 110,000 and up) (Supplementary
Figure S1). In the SWISS-2DPAGE, a double chain of 19 spots (pI/Mw: 6.65–7.78/63–67,000)
is presented [
22
]. The protein can be heavily glycosylated (12 N-linked annotations at 3 sites,
43 O-linked annotations at 34 sites) and phosphorylated (https://www.glygen.org/protein/
P02671 accessed on 10 September 2022).
2.44. FIBRINOGEN BETA CHAIN (FIBB_HUMAN)
The two-dimensional electrophoresis-pattern of FBB represents a chain of spots
(pI/Mw: 5.5–8.5/~52,000) (Supplementary Figure S1). In the SWISS-2DPAGE, a chain
of four spots (pI/Mw: 6.1–6.55/55–56,000) is presented [
22
]. The protein can be glyco-
sylated (52 N-linked annotations at 4 sites and 5 O-linked annotations at 3 sites) (https:
//www.glygen.org/protein/P02675 accessed on 10 September 2022).
2.45. FIBRINOGEN GAMMA CHAIN (FIBG_HUMAN)
The two-dimensional electrophoresis-pattern of FGG represents a chain of spots with
pI 4.5–7 (Mw ~ 50,000) (Supplementary Figure S1). In the SWISS-2DPAGE, 3 chains of
13 spots (pI/Mw: 5.07–5.65/44–51,000) are presented [
22
]. The protein is glycosylated (39
N-linked annotations at 4 sites, 1 O-linked annotation at 1 site) and phosphorylated at S68
(https://www.glygen.org/protein/P02679 accessed on 10 September 2022).
2.46. FIBRONECTIN (FINC_HUMAN)
The two-dimensional electrophoresis pattern of fibronectin represents a chain of spots
with pI 4.5–6.7 (Mw ~ 112,000 up) (Supplementary Figure S1). The protein is heavily gly-
cosylated (265 N-linked annotations at 13 sites, 37 O-linked annotations at 25 sites) and
phosphorylated (https://www.glygen.org/protein/P02751 accessed on 10 September 2022).
2.47. PLASMA GELSOLIN (GELS_HUMAN)
The two-dimensional electrophoresis pattern of gelsolin represents a chain of spots
with pI 4.5–6.5 (Mw ~ 83,000) (Supplementary Figure S1). The protein can be heavily
phosphorylated (25 sites), acetylated (12 sites), and ubiquitinated (10 sites) (https://www.
phosphosite.org accessed on 10 September 2022).
2.48. GLUTATHION PEROXIDASE 3 (GPX3_HUMAN)
The two-dimensional electrophoresis pattern of this protein represents a chain of spots
with pI 4.9–6.9 (Mw ~ 25,000) (Supplementary Figure S1). It is phosphorylated (4 sites) and
acetylated (3 sites) (https://www.phosphosite.org/ accessed on 10 September 2022).
Int. J. Mol. Sci. 2022,23, 11113 16 of 35
2.49. HEMOGLOBIN SUBUNIT ALPHA (HBA_HUMAN)
The two-dimensional electrophoresis pattern of this protein represents a chain of spots
with pI 7.5–9 (Mw ~ 15,000) (Supplementary Figure S1). In the SWISS-2DPAGE, two spots
(pI/Mw: 9.2/11,000, 8.9/11,000) are presented. The protein is glycosylated (3 O-linked
annotations at 3 sites), glycated (6 sites) (https://www.glygen.org/protein/P69905 accessed
on 10 September 2022), phosphorylated (17 sites), acetylated (4 sites), and ubiquitinated
(8 sites) (https://www.phosphosite.org/ accessed on 10 September 2022).
2.50. HEMOGLOBIN SUBUNIT BETA (HBB_HUMAN)
The two-dimensional electrophoresis-pattern of this protein represents a chain of spots
with pI 6.5–6.9 (Mw ~ 15,000) (Supplementary Figure S1). In the SWISS-2DPAGE, two
spots (pI/Mw: 7/15,000, 6.9/15,000) are presented. The protein is glycosylated (4 O-linked
annotations at 4 sites), glycated (6 sites), and phosphorylated (14 sites) (https://www.glygen.
org/protein/P68871 accessed on 10 September 2022).
2.51. HEMOPEXIN (HEMO_HUMAN)
The two-dimensional electrophoresis pattern of this protein represents a chain of spots
with pI 5–6.9 (Mw ~ 50,000) (Supplementary Figure S1). In the SWISS-2DPAGE, chains of five
spots (pI 5.25–5.59/Mw ~ 72–77,000) and two spots (4.48/19,274, 4.56/18,289) are presented.
The protein is glycosylated (184 N-linked annotations at 6 sites, 21 O-linked annotations at
6 sites (https://www.glygen.org/protein/P02790 accessed on 10 September 2022)).
2.52. HEPARIN COFACTOR 2 (HEP2_HUMAN)
The two-dimensional electrophoresis-pattern of this protein represents a chain of spots
with pI 4.9–6.5 (Mw ~ 55,000) (Supplementary Figure S1). The protein is glycosylated
(39 N-linked annotations at 3 sites, 13 O-linked annotations at 9 sites) and phosphorylated
at S37 (https://www.glygen.org/protein/P05546 accessed on 10 September 2022).
2.53. HAPTOGLOBIN (HPT_HUMAN)
The two-dimensional electrophoresis pattern of Hp represents ~16 spots of beta chains
with pI 4.8–6.0 (Mw ~ 40,000) and 3 spots of alpha 2 chain (Figure 2). In the SWISS-2DPAGE,
a chain of 19 spots (pI 4.88–5.86/Mw ~ 40,000, beta chain), 3 spots (pI 5.68–6.37/Mw ~
17,000, alpha 2 chain), and 2 spots (pI 5.13–5.37/Mw ~ 12,000, alpha 1 chain) are presented.
Hp is heavily glycosylated (351 N-linked annotations at 4 sites, 1 O-linked annotations at
1 site) (https://www.glygen.org/protein/P00738 accessed on 10 September 2022).
2.54. HAPTOGLOBIN-RELATED PROTEIN (HPTR_HUMAN)
The two-dimensional electrophoresis pattern of this protein represents a chain of spots
with pI 4.6–6.5 (Mw ~ 40,000) (Supplementary Figure S1). The protein is N-linked glycosy-
lated (5 sites), acetylated (1 site), and ubiquitinated (2 sites) (https://www.phosphosite.org/
accessed on 10 September 2022).
2.55. HISTIDINE-RICH GLYCOPROTEIN (HRG_HUMAN)
The two-dimensional electrophoresis-pattern of this protein represents a chain of
spots with pI 4.5–7.8 (Mw ~ 64,000) and spots around pI/Mw: 5.5/53,000 (Supplementary
Figure S1). In the SWISS-2DPAGE, only a single spot (pI/Mw: 5.3/53,000) is present.
The protein is glycosylated (44 N-linked glycans at 4 sites, 4 O-linked glycans at 3 sites)
(https://www.glygen.org/protein/P04196 accessed on 10 September 2022).
Int. J. Mol. Sci. 2022,23, 11113 17 of 35
Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 17 of 17
(a)
(b)
(c)
(d)
Figure 2. Two-dimensional electrophoresis patterns of haptoglobin alpha and beta chains. (a) A
sectional analysis of alpha and beta chains. The stained gel was divided into the sections with the
predetermined coordinates. Then each section was treated and analyzed by LC ESI-MS/MS (see
Materials and Methods, Section 4.2, 2DE). According to the abundance (emPAI) of Hpt in each
section, the graph was plotted. (b) A semi-virtual 2DE of alpha and beta chains (see Materials and
Methods, Section 4.2, 2DE). According to the emPAI of alpha chain (theoretical Mw: 15,946) or beta
chain (theoretical Mw: 27,265) in the sections, the graph was plotted. The ball size is proportional to
emPAI in each section. (c) Two-dimensional electrophoresis–Western of beta chain. (d) Two-
dimensional electrophoresis–Western of alpha2 chain.
1.
Figure 2.
Two-dimensional electrophoresis patterns of haptoglobin alpha and beta chains. (
a
) A
sectional analysis of alpha and beta chains. The stained gel was divided into the sections with the
predetermined coordinates. Then each section was treated and analyzed by LC ESI-MS/MS (see
Materials and Methods, Section 4.2, 2DE). According to the abundance (emPAI) of Hpt in each section,
the graph was plotted. (
b
) A semi-virtual 2DE of alpha and beta chains (see Materials and Methods,
Section 4.2, 2DE). According to the emPAI of alpha chain (theoretical Mw: 15,946) or beta chain
(theoretical Mw: 27,265) in the sections, the graph was plotted. The ball size is proportional to emPAI
in each section. (
c
) Two-dimensional electrophoresis–Western of beta chain. (
d
) Two-dimensional
electrophoresis–Western of alpha2 chain.
2.56. PLASMA PROTEASE C1 INHIBITOR (IC1_HUMAN)
The two-dimensional electrophoresis pattern of this protein represents a long chain
of spots with pI 3.2–5.2 (Mw ~ 64,000). The protein is heavily glycosylated (107 N-linked
annotations at 8 sites, 33 O-linked annotations at 21 sites) (https://www.glygen.org/
protein/P05155 accessed on 10 September 2022).
2.57. INTER-ALPHA-TRYPSIN INHIBITOR HEAVY CHAINS (ITIH1, ITIH2, ITIH3, ITIH4,
ITIH5)
In our experiments, 2DE patterns of these proteins are presented by the chains of the
precursor proteoforms and the mature ITIH1 (Supplementary Figure S1). The proteins
are heavily glycosylated, phosphorylated, acetylated, and ubiquitinated (https://www.
phosphosite.org/ accessed on 10 September 2022).
2.58. KALLISTATIN (KAIN_HUMAN)
In our experiments, the 2DE pattern of this protein is presented as a cluster of proteo-
forms around pI/Mw: 6–7/40–120,000 (Supplementary Figure S1). The protein is glycosy-
lated (31 N-linked annotations at 4 sites) and phosphorylated (https://www.glygen.org/
Int. J. Mol. Sci. 2022,23, 11113 18 of 35
protein/P29622 accessed on 10 September 2022, https://www.phosphosite.org/ accessed
on 10 September 2022.
2.59. KININOGEN 1 (KNG1_HUMAN)
The two-dimensional electrophoresis pattern of this protein represents multiple spots
(pI 3.5–8.5, Mw ~ 35–64,000) (Supplementary Figure S1). In the SWISS-2DPAGE, only
a single spot (pI/Mw: 6.48/7490) is present. The protein can be heavily glycosylated
(159 N-linked annotations at 6 sites, 65 O-linked annotations at 26 sites) (https://www.
glygen.org/protein/P01042 accessed on 10 September 2022), phosphorylated, acetylated,
and ubiquitinated (https://www.phosphosite.org/ accessed on 10 September 2022).
2.60. PHOSPHATIDYLCHOLINE-STEROL ACYLTRANSFERASE (LCAT_HUMAN)
The two-dimensional electrophoresis pattern of this protein represents three spots
(pI 4.0–4.8, Mw ~ 50,000) (Supplementary Figure S1). The protein is glycosylated (22 N-
linked annotations at 5 sites, 3 O-linked annotations at 3 sites) (https://www.glygen.
org/protein/P04180 accessed on 10 September 2022), phosphorylated, and ubiquitinated
(https://www.phosphosite.org/ accessed on 10 September 2022).
2.61. LUMICAN (LUM_HUMAN)
The two-dimensional electrophoresis pattern of this protein represents chains of spots
(pI 4.5–6.5, Mw ~ 52–83,000) (Supplementary Figure S1). The protein has multiple PTMs:
(97 N-linked glycans at 4 sites, 2 O-linked glycans at 3 sites, phosphorylation (11 sites),
and acetylation (8 sites) (https://www.uniprot.org/uniprotkb/P51884/entry accessed on
10 September 2022).
2.62. MANNOSE-BINDING PROTEIN C (MBL2_HUMAN)
The two-dimensional electrophoresis pattern of this protein represents just a single
spot (pI/Mw: 5.3/26,000) (Supplementary Figure S1).
2.63. PIGMENT EPITHELIUM-DERIVED FACTOR (PEDF_HUMAN)
The two-dimensional electrophoresis-pattern of this protein represents a chain of
spots (pI 4.5–6.5, Mw ~ 40–52,000) (Supplementary Figure S1). The protein is glycosylated
(10 N-linked glycans at 1 site, 2 O-linked glycans at 5 sites), phosphorylated (10 sites),
acetylated, and methylated (https://www.uniprot.org/uniprotkb/P36955/entry accessed
on 10 September 2022).
2.64. N-ACETYLMURAMOYL-L-ALANINE AMIDASE (PGRP2_HUMAN)
The two-dimensional electrophoresis pattern of this protein represents a chain of
spots (pI 5.5–6.8, Mw ~ 52–64,000) (Supplementary Figure S1). The protein is glycosylated
(12 N-linked glycans at 3 sites, 4 O-linked glycans at 7 sites) and phosphorylated (4 sites)
(https://www.uniprot.org/uniprotkb/Q96PD5/entry accessed on 10 September 2022).
2.65. PHOSPHATIDYLINOSITOL-GLYCAN-SPECIFIC PHOSPHOLIPASE D
(PHLD_HUMAN)
The two-dimensional electrophoresis pattern of this protein represents a chain of spots
(pI 4.2–5.6, Mw ~ 83–116,000) (Supplementary Figure S1). The protein is glycosylated
(10 sites, 22 N-linked glycans at 4 sites), phosphorylated (5 sites), and acetylated (https:
//www.phosphosite.org/ accessed on 10 September 2022).
2.66. PLASMINOGEN (PLMN_HUMAN)
The two-dimensional electrophoresis pattern of this protein represents two chains of
multiple spots (pI 3.3–4.1, Mw ~ 83–116,000) and (pI 6.7–8.5, Mw ~ 83–116,000)
(Supplementary Figure S1). In the SWISS-2DPAGE, a single chain (7 spots) is present
(pI 6.32–6.49, Mw ~ 112–116,000). The protein is glycosylated (54 N-linked glycans at 4
Int. J. Mol. Sci. 2022,23, 11113 19 of 35
sites, 12 O-linked glycans at 12 sites) and phosphorylated (15 sites) (https://www.uniprot.
org/uniprotkb/P00747/entry accessed on 10 September 2022).
2.67. PARAOXONASE (PON1_HUMAN)
The two-dimensional electrophoresis pattern of this protein represents a cluster of
spots (pI 4.0–5.5, Mw ~ 35–52,000) (Supplementary Figure S1). In the SWISS-2DPAGE,
two spots are presented (pI/Mw: 4.84/45,937 and 4.93/43,391). The protein is glycosylated
(30 N-linked glycans at 3 sites), phosphorylated (3 sites), and acetylated (1 site) (https:
//www.uniprot.org/uniprotkb/P27169/entry accessed on 10 September 2022).
2.68. PROPERDIN (PROP_HUMAN)
The two-dimensional electrophoresis pattern of this protein represents two spots
(pI/Mw: 8.5/52,000 and 8.7/52,000) (Supplementary Figure S1). The protein is glycosylated
(15 C-linked annotations at 15 sites, 2 N-linked annotations at 1 site, 4 O-linked annotations
at 4 sites).
2.69. VITAMIN K-DEPENDENT PROTEIN S (PROS_HUMAN)
The two-dimensional electrophoresis pattern of this protein represents a chain of spots
(pI 3.5–4.5, Mw ~ 64–83,000) (Supplementary Figure S1). The protein is glycosylated (5 N-
linked annotations at 3 sites, 4 O-linked annotations at 4 sites) and phosphorylated (8 sites)
(https://www.uniprot.org/uniprotkb/P07225/entry accessed on 10 September 2022).
2.70. PLASMA RETINOL-BINDING PROTEIN (RET4_HUMAN)
The two-dimensional electrophoresis pattern of this protein represents a cluster of
spots (pI 5.0–6.0, Mw ~ 18–26,000) (Supplementary Figure S1). In the SWISS-2DPAGE,
three spots are presented (pI ~ 5.0, Mw ~ 20,000). The protein can be phosphorylated and
methylated (https://www.phosphosite.org/ accessed on 10 September 2022).
2.71. SERUM AMYLOID A (SAA1_HUMAN)
The two-dimensional electrophoresis pattern of this protein represents two spots
(pI/Mw: ~5.6/12,000 and ~5.8/12,000) (Supplementary Figure S1). This protein is phos-
phorylated (5 sites) (https://www.phosphosite.org/ accessed on 10 September 2022).
2.72. SERUM AMYLOID P (SAMP_HUMAN)
The two-dimensional electrophoresis pattern of this protein represents a cluster of
spots (pI/Mw: ~4.5–6.1/21–35,000) (Supplementary Figure S1). This protein is phosphory-
lated (7 sites), acetylated (3 sites), ubiquitinated (2 sites) (https://www.phosphosite.org/
accessed on 10 September 2022), and glycosylated (14 N-linked glycans at 1 site, 1 O-linked
glycan) (https://www.glygen.org/protein/P02743 accessed on 10 September 2022).
2.73. SEX HORMONE-BINDING GLOBULIN (SHBG_HUMAN)
The two-dimensional electrophoresis pattern of this protein represents a chain of spots
(pI/Mw: ~5.0–6.0/35–52,000) (Supplementary Figure S1). This protein is phosphorylated
(4 sites) (https://www.phosphosite.org/ accessed on 10 September 2022) and glycosylated
(12 N-linked glycans at 3 sites, 6 O-linked glycans at 1 site).
2.74. S100A8 (CALPOTECTIN) (S10A8_HUMAN)
The two-dimensional electrophoresis pattern of this protein represents one spot (pI/Mw:
~6.8/10,000) (Supplementary Figure S1). However, this protein can be heavily phosphorylated
(9 sites), acetylated (https://www.phosphosite.org/ accessed on 10 September 2022), and
glycosylated (1 O-linked glycan) (https://www.uniprot.org/ accessed on 10 September 2022).
Int. J. Mol. Sci. 2022,23, 11113 20 of 35
2.75. S100A9 (CALPOTECTIN) (S10A9_HUMAN)
The two-dimensional electrophoresis pattern of S100-A9 represents one spot (pI/Mw:
~6.0/10,000) (Supplementary Figure S1). This protein can be phosphorylated (5 sites),
acetylated (3 sites), methylated (https://www.phosphosite.org/ accessed on 10 Septem-
ber 2022), and glycosylated (1 O-linked glycan) (https://www.uniprot.org/ accessed on
10 September 2022).
2.76. TETRANECTIN (TETN_HUMAN)
The two-dimensional electrophoresis pattern of this protein represents a chain of spots
(pI/Mw: ~5–6.5/21–26,000) (Supplementary Figure S1). This protein can be glycosylated
(1 O-linked annotation(s) at 1 site) (https://www.uniprot.org/uniprotkb/P05452/entry
accessed on 10 September 2022).
2.77. THYROXINE-BINDING GLOBULIN (THBG_HUMAN)
The two-dimensional electrophoresis pattern of this protein represents a chain of spots
(pI/Mw: ~5–5.5/52–64,000) (Supplementary Figure S1). This protein can be glycosylated
(17 N-linked glycans at 3 sites, 1 O-linked glycan) and phosphorylated (https://www.
phosphosite.org/ accessed on 10 September 2022).
2.78. PROTHROMBIN (THRB_HUMAN)
The two-dimensional electrophoresis pattern of this protein represents a chain of spots
(pI/Mw: 5–6.5/64–83,000) (Supplementary Figure S1). In the SWISS-2DPAGE, there is a
chain of five spots (pI/Mw: 4.95–5.05/80,000). This protein can be glycosylated (2 N-linked
glycans at 5 sites, 1 O-linked glycan at 6 sites), phosphorylated (9 sites), acetylated (1 site),
and ubiquitinated (4 sites) (https://www.uniprot.org/uniprotkb/P00734/entry accessed
on 10 September 2022).
2.79. SEROTRANSFERRIN (TRFE_HUMAN)
The two-dimensional electrophoresis pattern of this protein represents a cluster of
spots (pI/Mw: ~5.7–7.0/64–83,000) (Supplementary Figure S1). In the SWISS-2DPAGE,
there are 3 chains of 22 spots (pI/Mw: 6.14–6.64/76–87,000) for serotransferrin. This
protein can be glycosylated (145 N-linked glycans at 6 sites, 6 O-linked glycans at 2 sites),
phosphorylated (21 sites), acetylated (10 sites), and ubiquitinated (7 sites) (https://www.
phosphosite.org/ accessed on 10 September 2022).
2.80. TRANSTHYRETIN (TTHY_HUMAN)
The two-dimensional electrophoresis pattern of this protein represents a chain of spots
(pI/Mw: 4.8–5.7/15–18,000) (Supplementary Figure S1). In the SWISS-2DPAGE, there are
a chain of three spots (pI/Mw: 5.02–5.52/13,800), and a spot (pI/Mw: 5.52/35,391) for
transthyretin. This protein can be glycosylated (1 N-linked annotation), phosphorylated
(6 sites), acetylated (2 sites), or ubiquitinated (4 sites) (https://www.phosphosite.org/
accessed on 10 September 2022).
2.81. VITAMIN D-BINDING PROTEIN (VTDB_HUMAN)
The two-dimensional electrophoresis pattern of this protein represents a cluster of
spots (pI/Mw: 4.5–5.7/40–52,000) (Supplementary Figure S1). In the SWISS-2DPAGE, there
are two spots (pI/Mw: 5.16/53,772 and 5.24/53,918) for vitamin D-binding protein. This
protein can be glycosylated (1 N-Linked glycan at 1 site, 1 O-Linked glycan), phosphory-
lated (12 sites), acetylated (1 site), or ubiquitinated (1 site) (https://www.phosphosite.org/
accessed on 10 September 2022).
2.82. VITRONECTIN (VTNC_HUMAN)
The two-dimensional electrophoresis pattern of this protein represents a cluster of
spots (pI/Mw: 3.7–6.6/52–116,000) (Supplementary Figure S1). In the SWISS-2DPAGE,
Int. J. Mol. Sci. 2022,23, 11113 21 of 35
there is only one spot (pI/Mw: 4.58/9248) for vitronectin. VN can be glycosylated (1 N-
linked annotation), phosphorylated (20 sites), acetylated (1 site), and ubiquitinated (1 site)
(https://www.phosphosite.org/ accessed on 10 September 2022).
2.83. ZINC-ALPHA-2-GLYCOPROTEIN (ZA2G_HUMAN)
The two-dimensional electrophoresis pattern of this protein represents a cluster of
spots (pI/Mw: 4.2–5.0/35–40,000) (Supplementary Figure S1). In the SWISS-2DPAGE,
there is a chain of four spots (pI/Mw: 4.8–4.97/40–42,000) for zinc-alpha-2-glycoprotein.
This protein can be glycosylated (108 N-Linked glycans at 3 sites, 1 O-Linked glycan at
1 site), phosphorylated (1 site), and ubiquitinated (5 sites) (https://www.phosphosite.org/
accessed on 10 September 2022).
3. Discussion
Tumorigenesis leads to multiple variations in the human plasma proteome that can be
dynamic and alterable during the progress of the disease. Practically all major, so-called
“classical”, plasma proteins change abundances or PTMs. The majority of these proteins
are secreted by the liver, so it could be anticipated to see these changes only in the case of
liver cancer. However, they can be observed with other tumors as well as cancer induces
disturbances in the blood homeostasis that is supported by “classical plasma proteins”. It
follows that it is possible to search the specific/unspecific ways of tumor prediction not
only through the detection of products of the tumor but also by analyzing the changes
in “classic plasma proteins”. It is relevant to mention that plasma analysis by a very
different approach, differential scanning calorimetry (DSC), can give us a hint. Typically,
DSC is used to determine the partial heat capacity of macromolecules as a function of
temperature, from which their structural stability during thermal denaturation can be
assessed. The method is very sensitive and allows precise determination of thermally-
induced conformational transitions of proteins present in plasma. There are already quite
a few publications showing that DSC can be used to distinguish between normal and
cancerous plasma samples [
273
,
274
]. Moreover, the data obtained by this method can be
reproduced using major plasma proteins.
It follows that there is a possibility of building test systems based on these major
(“classical”) proteins. What is important is that many examples of such systems have
been introduced already. For example, the relationship between inflammation and clinical
outcome is described using the Modified Glasgow Prognostic Scale (mGPS), which includes
levels of C-reactive protein (CRP) and albumin [
275
]. The combination of elevated CRP
(>10 mg/L) and decreased albumin (<35 g/L) corresponds to higher mGPS, which corre-
lates with systemic inflammation and poor outcome of cancer therapy [
276
]. The OVA1 test
uses the other major plasma proteins. OVA1 is an FDA-approved blood test that measures
the levels of five proteins (CA125, transferrin, transthyretin, apolipoprotein A1, and beta-2
microglobulin) to detect ovarian cancer risk in women. Here, a sophisticated mathematical
formula (multivariate index assay) is used to evaluate and combine the levels of these
proteins in plasma, producing an ovarian cancer risk score. Using this approach, OVA1
can detect early-stage ovarian cancer with 98% specificity. The OVERA (second-generation
or OVA2) assesses a woman’s malignancy risk using combined results from the following
five proteins: apolipoprotein A1, human epididymis protein 4 (HE4), CA-125 II, follicle-
stimulating hormone (FSH), and transferrin (Vermillion Inc. OVA1 Products. Updated
2020. Available at: https://vermillion.com/ova-products accessed on 10 September 2022).
The observation of enhanced levels of clusterin, ITIH4, antithrombin-III, and C1RL in sera
of endometrial cancer patients allowed a mathematical model to be built to detect cancer
samples [
29
]. Accordingly, by the selection of the appropriate panels (proteomics signa-
tures) of the plasma oncomarkers, it is possible to detect/monitor different types of cancers.
The main point is to select the correct set of oncomarkers and develop an algorithm that
will take into account all possible changes in these oncomarkers (level, PTMs etc.) that are
related to cancer. This selection should be meticulously performed based on oncomarker
Int. J. Mol. Sci. 2022,23, 11113 22 of 35
behavior in plasma, not in tissue. We performed a search for publications with information
(level, PTMs) about “classical” plasma proteins in the case of malignant processes in the
human body (Table 1). As levels of some oncomarkers behave differently in different
cancers (rise or fall), the test could specifically detect the type of cancer. Apolipoproteins
are a good example here. SAA1 and CRP are APPs that are routinely measured in the
clinic. The level of apoA-1 is reduced in many cancers but increased in some [
80
]. The
decreased level of apoA-I in plasma is observed in the case of de novo myelodysplastic
syndromes [
83
], NSCLC [
84
], nasopharyngeal carcinoma (NPC) [
85
], esophageal squamous
cell carcinoma [
86
], and BC [
75
], but it is increased in SCLC, HCC, and bladder cancer [
80
].
The level of apoA-II is dramatically reduced in the serum of patients with gastric cancer
and multiple myeloma [
70
,
87
] but increased in HCC and prostate cancer [
88
,
89
]. A similar
situation can be observed for other apolipoproteins [80].
Another aspect that should be considered is the appearance of proteoforms produced
by genetic polymorphisms, alternative splicing, PTMs, etc. These events change the charge
(pI) and the weight (Mw) of the protein. Because of that, the experimental pI/Mw of the
proteins can be different from the theoretical ones. This leads to the production of sets of
proteoforms that in our case are detected as 2DE patterns. There is a belief that some 2DE
patterns can be different between norm and cancer and could be used as specific biomarkers.
Thus far, there are not many such examples, but progress in proteomics methods should
improve the situation [
277
,
278
]. Proteomics is generating and analyzing a large volume of
data and these data exactly fit the situation with multiple variations in plasma proteomes
during cancer development and progression. Here, high-throughput, quantitative mass
spectrometry is the best choice. There is already a good example of the possibility of using
it in the clinic [
14
]. Geyer et al. introduced a rapid and robust “plasma proteome profiling”
LC-MS/MS pipeline. Their single-run shotgun proteomics workflow enables quantitative
analysis of hundreds of plasma proteins from just 1 µL of plasma [14].
Our aim is to build a comprehensive proteoform database containing norm and
cancer samples http://2de-pattern.pnpi.nrcki.ru/ accessed on 10 September 2022 [
30
].
Glioblastoma and hepatocellular carcinoma are the cancers in our study so far. The database
contains only the cellular samples, but we are in the process of incorporating tissue and
plasma samples.
4. Materials and Methods
4.1. Plasma
The pooled human plasma was from healthy male donors (age 20–47 years) [
278
,
279
].
Depletion of serum albumin and immunoglobulins IgG was carried out according to
Agilent Multiple Affinity Removal System (MARS) protocol (“Agilent Technologies”, Santa
Clara, CA, USA) [280,281].
4.2. Two-Dimensional Electrophoresis
The detailed process was described previously [
280
]. In short, 10
µ
L of plasma (0.5 mg
of protein) was mixed with 20
µ
L of lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS, 1%
DTT, 2% (v/v) ampholytes, pH 3–10, protease inhibitor cocktail) and then with 100
µ
L of
rehydrating buffer (7 M urea, 2 M thiourea, 2% CHAPS, 0.3% DTT, 0.5% IPG (v/v) buffer,
pH 3–11 NL, 0.001% bromophenol blue). Immobiline DryStrip 3–11 NL (7 cm) was passively
rehydrated by this solution for 4 h at 4
◦
C. IEF was run on Hoefer
™
IEF100 (“Thermo Fisher
Scientific”, Waltham, MA, USA). After IEF, strips were incubated 10 min in the equilibration
solution (50 mM Tris, pH 8.8, 6 M urea, 2% SDS, 30% (v/v) glycerol, 1% DTT), following in
the same solution with 5% IAM instead of DTT. The strips were sealed with a hot solution
of 0.5% agarose prepared in electrode buffer (25 mM Tris, pH 8.3, 200 mM glycine, and
0.1% SDS) on top of the polyacrylamide gel (14%), and run in the second direction [
280
].
Gels stained by Coomassie Blue R350 were scanned by ImageScanner III and analyzed
using Image Master 2D Platinum 7.0. For the sectional 2DE analysis, this gel was cut into
96 sections with determined coordinates. Each section (~0.7 cm
2
) was shredded and treated
Int. J. Mol. Sci. 2022,23, 11113 23 of 35
with trypsin. Tryptic peptides were eluted from the gel by extraction solution (5% (v/v)
ACN, 5% (v/v) formic acid) and dried in Speed Vac. In the case of a semi-virtual 2DE, the
18-cm Immobiline DryStrip 3–11 NL was cut into 36 equal sections after IEF. For complete
reduction, 300
µ
L of 3 mM DTT and 100 mM ammonium bicarbonate were added to each
section and incubated at 50
◦
C for 15 min. For alkylation, 20
µ
L of 100 mM IAM were
added and samples were incubated in the dark at r.t. for 15 min. The peptides were eluted
with 60% acetonitrile and 0.1% TFA and dried in Speed Vac.
4.3. ESI LC-MS/MS Analysis
A detailed procedure was described previously [
279
,
280
]. Peptides were dissolved
in 5% (v/v) formic acid. Tandem mass spectrometry analysis was conducted in duplicate
on an Orbitrap Q-Exactive mass spectrometer (“Thermo Fisher Scientific”, Waltham, MA,
USA). The data were analyzed by Mascot “2.4.1” (“Matrix Sciences”, Mount Prospect, IL,
USA) or SearchGui [
282
] using the following parameters: enzyme—trypsin; maximum of
missed cleavage sites—2; fixed modifications—carbamidomethylation of cysteine; variable
modifications—oxidation of methionine, phosphorylation of serine, threonine, tryptophan,
acetylation of lysine; the precursor mass error—10 ppm; the product mass error—0.01 Da.
As a protein sequence database, UniProt (October 2014) was used.
Only 100% confident results of protein identification were selected. Two unique
peptides per protein were required for all protein identifications. Exponentially modified
PAI (emPAI), the exponential form of protein abundance index (PAI) defined as the number
of identified peptides divided by the number of theoretically observable tryptic peptides
for each protein, was used to estimate protein abundance [283].
4.4. Immunostaining (Western Blotting)
Plasma proteins (0.5 mg) were run by 2DE (cm 2DE, using 13-cm strip pH 4–7). Pro-
teins were transferred (2 h, 28 V) from the gel onto PVDF membrane (Hybond P, 0.2
µ
m)
using two sheets of thick paper (Bio-Rad, Hercules, CA, USA), saturated with 48 mM Tris,
39 mM glycine, 0.037% SDS, 20% ethanol. The membrane was treated following a protocol
of Blue Dry Western [
36
] and treated with antibodies [
21
]. Primary antibodies were mouse
monoclonal anti-Hp (C8, sc-376893, or F8, sc-390962, from “Santa Cruz Biotechnology”,
Santa Cruz, CA, USA) in dilution 1/25 (80 ng/mL in TBS (25 mM), Tris (pH 7.5) and
150 mM NaCl containing 3% (w/v) BSA) or rabbit polyclonal anti-Hp (MBS177476, My-
BioSource, San Diego, CA, USA). Secondary goat anti-mouse immunoglobulins G labeled
by horseradish peroxidase (NA931V, “GE Healthcare”, Chicago, IL, USA ) were used in
TBS containing 3% (w/v) nonfat dry milk (1/5000 dilution). The reaction was developed
using ECL (Western Lightning Ultra, “PerkinElmer”, Waltham, MA, USA) and X-ray film
(Amersham Hyper film ECL).
5. Conclusions
For now, proteomics is collecting big data about the human plasma proteome http:
//plasmaproteomedatabase.org/index.html accessed on 10 September 2022 [
284
]. These
data include many proteome parameters: their dynamics, different protein presence, abun-
dance, modifications, variations, etc. In the case of cancer, a proteome performs multiple
perturbations, where all its components are involved through changes in their levels and
modifications. Here, the plasma proteome works as a united entity that executes and
reflects the processes in the human body. Accordingly, the profiling of plasma proteomes
is a promising and powerful approach to follow these processes. This profiling could
combine hundreds of already known plasma biomarkers and has a very promising future
in biomedicine as it could disclose information about any abnormal situation in the human
body including cancer. There is a big chance that MS-based proteomics will become a
part of the routine medical technique [
14
,
285
]. In addition to the usual MS analysis of
proteins/proteoforms, this technique should include special processing programs allow-
ing conclusions to be made about the human body’s state based on these variations in
Int. J. Mol. Sci. 2022,23, 11113 24 of 35
protein/proteoform signatures/profiles (level, PTMs, etc.). In our work, we collected
information about the connection of cancers with levels of “classical plasma proteins”
and generated their proteoform profiles (Table 1, Supplementary Figure S1). As a next
step, similar profiles representing protein perturbations in plasma produced in the case of
different cancers should be generated. Moreover, based on this information, different test
systems can be developed.
Supplementary Materials:
The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/ijms231911113/s1.
Author Contributions:
Conceptualization, S.N.; validation, N.R., E.Z. and V.Z.; formal analysis, N.R.,
E.Z., N.K., F.K. and V.Z.; investigation, N.R., E.Z. and V.Z.; data curation, N.R., E.Z., N.K. and V.Z.;
writing, S.N.; visualization, N.R. and E.Z.; supervision, S.N.; project administration, S.N. All authors
have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement:
The study was conducted according to the guidelines of
the Declaration of Helsinki, and approved by the Local Ethics Committee of Petersburg Institute of
Nuclear Physics (PNPI) of National Research Center “Kurchatov Institute” (protocol code 02_2020
from 21 April 2020).
Informed Consent Statement:
Informed consent was obtained from all subjects involved in the study.
Data Availability Statement: Not applicable.
Acknowledgments:
The work was performed within the framework of the Program for Basic Re-
search in the Russian Federation for a long-term period (2021–2030) (
№
122030100168-2). Mass
spectrometry measurements were performed using the equipment of the “Human Proteome” Core
Facilities of the Institute of Biomedical Chemistry (Moscow, Russia).
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
MS Mass spectrometry
ESI LC-MS/MS Liquid chromatography–electrospray ionization tandem mass spectrometry
GBM Glioblastoma multiform
2DE Two-dimensional gel electrophoresis
emPAI Exponentially modified protein abundance index
HCC Hepatocellular carcinoma
CRC Colorectal cancer
NSCLC Non-small cell lung cancer
SCLC Small cell lung carcinoma
HDL High-density lipoproteins
cSCC Cutaneous squamous cell carcinoma
BC Breast cancer
OC Ovarian cancer
PDAC Pancreatic cancer
OSCC Oral squamous cell carcinoma
GC Gastric cancer
MM Multiple myeloma
PC Prostate cancer
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