Finding one's way in proteomics: a protein species nomenclature.

Abstract

Our knowledge of proteins has greatly improved in recent years, driven by new technologies in the fields of molecular biology and proteome research. It has become clear that from a single gene not only one single gene product but many different ones - termed protein species - are generated, all of which may be associated with different functions. Nonetheless, an unambiguous nomenclature for describing individual protein species is still lacking. With the present paper we therefore propose a systematic nomenclature for the comprehensive description of protein species. The protein species nomenclature is flexible and adaptable to every level of knowledge and of experimental data in accordance with the exact chemical composition of individual protein species. As a minimum description the entry name (gene name + species according to the UniProt knowledgebase) can be used, if no analytical data about the target protein species are available.

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Chemistry Central Journal
Open Access
Commentary
Finding one's way in proteomics: a protein species nomenclature
Hartmut Schlüter*1, Rolf Apweiler2, Hermann-Georg Holzhütter3 and
Peter R Jungblut*4
Address: 1University Medical Centre Hamburg-Eppendorf, Hamburg, Germany, 2The EMBL Outstation-The European Bioinformatics Institute,
Wellcome Trust Genome Campus, Hinxton, Cambridge, Great Britain, 3Charité Berlin, Institut für Biochemie, Berlin, Germany and 4Max Planck
Institute for Infection Biology, Berlin, Germany
Email: Hartmut Schlüter* - hschluet@uke.de; Rolf Apweiler - apweiler@ebi.ac.uk; Hermann-Georg Holzhütter - hergo@charite.de;
Peter R Jungblut* - jungblut@mpiib-berlin.mpg.de
* Corresponding authors
Abstract
Our knowledge of proteins has greatly improved in recent years, driven by new technologies in the
fields of molecular biology and proteome research. It has become clear that from a single gene not
only one single gene product but many different ones - termed protein species - are generated, all
of which may be associated with different functions. Nonetheless, an unambiguous nomenclature
for describing individual protein species is still lacking. With the present paper we therefore
propose a systematic nomenclature for the comprehensive description of protein species. The
protein species nomenclature is flexible and adaptable to every level of knowledge and of
experimental data in accordance with the exact chemical composition of individual protein species.
As a minimum description the entry name (gene name + species according to the UniProt
knowledgebase) can be used, if no analytical data about the target protein species are available.
Introduction
The number of publications in the field of proteomics has
increased dramatically over the last decade. The driving
force behind this development has been the hope of gain-
ing additional insights into the functioning of a cell or of
a complete organism by identification and quantification
of proteins in different biological states such as disease
and health, wild type and mutant, baseline and perturbed
state, among others. The dynamics and the influence of
post-translational protein modifications were mostly
ignored in the course of development of the basic technol-
ogies during these years. The focus on technology has
nonetheless resulted in dramatic improvements in mass
spectrometry and in coupling MS with two-dimensional
electrophoresis and liquid chromatography. The improve-
ments and results gained over time in proteomics research
have shown that the behaviour and variability of proteins
are more complex than had ever been imagined. The fact
that the same protein was found at several different spots
on 2-dimensional electrophoresis gels made it necessary
to define a new term for these different forms of a single
protein: protein species [1,2]. Each additional modifica-
tion and each new combination of modifications repre-
sents an additional protein species of that single protein.
Though the term protein species had been used earlier in
the literature [3,4], it was not clearly defined and used
more in the sense of a protein complex consisting of sev-
eral subunits [3] or to differentiate between different pro-
teins (e.g. catalase and actin were two different protein
species) [4]. According to the IUPAC rules [5] the term
"isoform" is to be used for genetic variations such as
allelic forms. Therefore, it was necessary to find a term for
any chemical modification and any combination of
chemical modifications. The term "protein species" has
been defined by Jungblut et al. at the chemical, molecular
level [1,2]. According to this definition, isoforms repre-
Published: 9 September 2009
Chemistry Central Journal 2009, 3:11doi:10.1186/1752-153X-3-11
Received: 8 June 2009
Accepted: 9 September 2009
This article is available from: http://journal.chemistrycentral.com/content/3/1/11
© 2009 Schlüter et al

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sent different protein species, because they are also chem-
ically different. In contrast, two proteins with different
post-translational modifications represent different pro-
tein species but not different isoforms.
About 600 different post-translational modifications
(PTMs) are included in the database UNIMOD (August,
2009, [6]), which was developed by Creasy and Cottrell
[7]. Based on the results they obtained in a proteome ana-
lytical study employing high-resolution Fourier-Trans-
form-Ion-Cyclotron mass spectrometry (FT-ICR-MS)
Nielsen et al. concluded that the estimated level of 8-12
modified peptides per unmodified tryptic peptide present
at >1% level approaches one modification per amino acid
on average [8]. An example for the important relationship
between the exact chemical composition of proteins
including PTMs and their function is the polyubiquiti-
nylation of proteins. Lysin-48-linked poly-ubiquitin
chains target proteins for proteasome-mediated proteoly-
sis, whereas lysine-63-linked ubiquitin chains mediate
various non-degradative functions, including the activa-
tion of signalling factors and protein trafficking [9]. To
move the field forward, it has become necessary, there-
fore, to take the speciation of proteins and the kinetics of
their protein species into account [2].
Discussion
Three main proteomic approaches
The classic strategy, the 2-DE/MS approach, starts with the
separation of the proteins by two-dimensional electro-
phoresis [4] followed by enzymatic digestion and identi-
fication of the proteins by mass spectrometric analysis of
the peptide digest (Figure 1, path 1). The comparison of
the mass spectrometric data with sequence databases
results in the identification of the protein. This approach
has the advantage of separation of the proteins at the pro-
tein species level with a high resolution of up to 10,000
spots [10] and application of the identification procedure
at the peptide level, where MS is very sensitive, fast and
accurate [11].
The second strategy (path 2 in Figure 1) starts directly with
the digestion of the proteins of a complex mixture. Low
femtomole protein identifications in mixtures by online
LC/MS/MS were first reported in 1997 [12]. The digestion
yields a huge number of peptides, which are separated in
the next step, typically via one-dimensional or multidi-
mensional chromatographic methods. This procedure is a
bottom-up approach because it starts on the level of sepa-
ration with peptides. Peptides eluting from a reversed-
phase column are then identified by mass spectrometry
The three main strategies in proteomics
Figure 1
The three main strategies in proteomics. 2-DE: two-dimensional electrophoresis; LC: liquid chromatography; n-D: n-
dimensional; db: database; MS: mass spectrometry; PMF: peptide mass fingerprint; PTM: post-translational modification.
Organism
Compartment
protein aa sequence + PTMs
2-DEn-D-LC
LC
digestion
protein species
MSMS/MS
peptide aa sequences
digestion
extremely complex
mixture of peptides
complex mixture
of peptides
Protein extract:
Complex mixture of protein species
LC-MS/MS
mixture of peptides
peptide aa sequences
+ PTM
PMF
protein species
db analysis db analysis
db analysis
Identity of the
protein species
Identity of the
protein species
Identity of the
protein-coding gene
Bottom-up approach
Top-down approach
MS + MS/MS
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(MS), complementing MS with MS/MS experiments. The
latter analysis yields amino acid sequences by which the
original proteins can be identified, again by sequence
database comparison. The bottom-up approach is fast and
sensitive, but does not allow the differentiation between
protein species.
A third strategy, a top-down approach (path 3 in Figure 1)
starts with liquid chromatography for separation of the
protein species followed by identification of the protein
species by mass spectrometry [13]. This approach, how-
ever, has been largely limited to low mass proteins up to
30 kDa, although there has been a report of identification
of a protein with a mass of more than 200 kDa [14]. With
the present technology the technique does afford a high
sample amount, protein fractions with a largely reduced
complexity of composition, and high mass accuracies in
the low ppm range. A top-down study in Hela cells
resulted in the identification of 45 protein species, con-
taining polymorphisms, alternative splicing products and
modifications [15].
In the bottom-up, top-down terminology the traditional
2-DE/MS approach represents a top-down separation with
bottom-up identification. The critical step in the first two
strategies is the protein digestion step, since usually not all
peptides of the digest are detected by mass spectrometric
analysis. During the separation steps peptides may be lost
due to unspecific interactions with surfaces and chroma-
tographic materials. Other peptides may also not be iden-
tified or cannot be used for the peptide mass
fingerprinting, if they contain uncommon or complex
post-translational modifications, or if they lie outside the
optimal mass range between 500 and 3000 Da. As a result,
the protein identification is in most cases based on a sub-
set of peptides and does not cover 100% of the amino acid
sequence of the analysed protein. A consequence of this is
that RNA and protein splice variants, proteolytically proc-
essed protein species, and protein polymorphisms cannot
be distinguished from each other. As a further conse-
quence, protein species containing post-translational
modifications may not be identified. This problem is
clearly reduced with the 2-DE strategy [16], since the pro-
tein species are separated and detected in spots. All the
peptides of one protein are within one mass spectrum,
permitting analysis of the modifications of the predicted
primary structure. Additionally, the sequence coverage
can be increased by using different digestion procedures.
In the pure bottom-up approach (strategy 2 in Figure 1)
the peptides derived from one protein species are distrib-
uted over all fractions of the LC, which makes it impossi-
ble to assign them to their respective protein species. Since
peptides with an identical amino acid sequence stemming
from different protein species elute within a single peak,
quantification of the individual protein species is not pos-
sible using this method.
Recently, an integrated top-down and bottom-up strategy
for broadly characterizing protein species has been devel-
oped to overcome the limitations of pure LC-MS strategies
[17]. Quantification and resolution of low-abundance
protein species still remain difficult problems for all of the
proteomic approaches used today.
How do gene polymorphisms, alternatively spliced
transcripts, proteolytically processed protein species, and
post-translational modifications affect the number of
protein species derived from a single gene?
Nucleotide polymorphisms, alternative splicing, and pro-
teolytic cleavage, as well as post-translational modifica-
tions, are widespread and have an obvious effect on the
number of protein species that can derive from a single
gene. There are no systematic data available on the distri-
bution of protein species derived from all genes in a single
organism, but evidence for the widespread occurrence of
multiple protein species per protein can be found in many
publications that present the results of 2-dimensional
electrophoresis (2-DE). One such report is by Scheler et al.
[18], where the authors presented a 2-DE pattern showing
59 spots of the Hsp27 protein. In another article, Klose et
al. [19] identified 24 protein spots encoded by the γ-eno-
lase-2 gene and 52 protein spots of the HSP-70 gene in
mouse brain tissue. Even in microorganisms, many differ-
ent protein species, particularly of the heat-shock proteins
such as HspX and GroES, have been found [20]. Better
known is the speciation in the case of histones and a spe-
cial histone code [21] has been postulated connecting cer-
tain histone species with defined functions. Twenty
modifications and all of their combinations result in a
total of more than 3 million protein species alone for his-
tone H4 [22]. The biological function of most of these
species is completely uncertain at present, but an exten-
sion of the histone code to other proteins has already been
discussed, and designated as the "protein code" [23].
How do polymorphisms, alternatively spliced transcripts,
proteolytically processed protein species, and
posttranslational modifications affect the protein
function?
Many examples of different protein species derived from
an initial synthesis product having different molecular
roles are now known. Reviews focusing on HSP-70 - the
example mentioned above - illustrate the involvement of
HSP-70 in many different cellular processes [24,25]. It can
be assumed that different protein species are responsible
for different cellular tasks. For the last twenty to thirty
years it has been well known that, for example, enzymatic
activities can be switched on and off by phosphorylation/
dephosphorylation processes. Protease-activated recep-

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tors (PAR), as well as many proteases, are activated by the
cleavage of a peptide from the protein by specific pro-
teases. The ESAT-6 gene product of Mycobacterium tubercu-
losis differentiates into at least 8 protein species [26]. In
this investigation, four of the ESAT-6 protein species were
acetylated at the N-terminus and were not able to interact
with CFP10, an interaction necessary for the transfer of
both proteins out of the bacterial cell. This observation of
CFP 10 binding and CFP 10 non-binding protein species
in the extra-cellular protein fraction raises new functional
questions.
The biological significance of the protein species concept
[1,2], which defines protein species chemically [2], is
clearer if we take a look at our current knowledge of gene
expression and the flow of information from genes to pro-
teins (Figure 2). In recent years it has become obvious that
the original paradigm of one gene encoding one protein is
not correct and must be replaced by a new one based on
the following facts:
1. At the end, one gene encodes many proteins - more
exactly, protein species - which are usually strongly inter-
related based on their amino acid sequence, but which
can, nonetheless, differ sometimes quite dramatically in
chemical structure as a result of alternative splicing at the
RNA and protein level [27] and/or due to post-transla-
tional modifications.
2. A protein species with a defined function is not only the
product of one gene but of many genes, since many other
products of completely different genes are involved in
processing the chemical structure of the mature protein
species.
3. Protein species are also modified by their environment,
e.g. by physical factors such as temperature or light, or
chemically by interactions with other molecules.
4. Furthermore, protein species interact with other pro-
teins encoded by their own genes, as well as with small
molecules. Figure 2 summarizes the new paradigm and
gives an overview of the flow of information.
Two examples of proteins present in the cell as different
protein species are the well-known proteins angiotensin-
converting-enzyme (ACE) (Figure 3) and glyceraldehyde-
3-phosphate dehydrogenase (GAPDH) (Figure 4). ACE is
a component of the renin-angiotensin system, which reg-
ulates blood pressure, fluid homeostasis, and electrolyte
balance. ACE contributes to blood pressure regulation by
generating the vasoactive octapeptide angiotensin II from
inactive decapeptide angiotensin I and by cleaving the
Scheme of the flow of information starting with extracellular events via receptors, transcription factors and other proteins reg- ulating gene expression and determining synthesis and processing of protein species
Figure 2
Scheme of the flow of information starting with extracellular events via receptors, transcription factors and
other proteins regulating gene expression and determining synthesis and processing of protein species. IP: inter-
acting partner biomolecules.
DNA
Initial
Protein
Species A
Protein
species A3
Protein
species A3
Protein
species An
Ribosome
PTMs
Protein
species A1
Protein
Species A1
Protein
species A3
RNA
Protein
Species A2
Environment
IP
m-RNA A
m-RNA B
Initial-
Protein
species B
Initial-
Protein
species Bn
Receptors, Regulators,
Transcription factors

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vasodilator bradykinin. ACE exists as at least two protein
species, germinal ACE (gACE) and somatic ACE (sACE),
which are coded by the same gene but differently tran-
scribed from two alternative tissue-specific promoters
[28]. The larger glycosylated protein species (150 - 170
kDa [29]), sACE, is synthesized in neuronal cells, macro-
phages, renal epithelial cells and vascular endothelial cells
and is involved in the regulation of blood pressure and
renal function. The smaller glycosylated form (100 - 110
kDa [29]), gACE, is synthesized in maturing sperm cells
and is involved in male fertility. Both protein species of
the ACE gene are located in the membrane of the cell with
a short cytoplasmic domain, a transmembrane domain,
and a long extracellular domain containing the active
sites. They are cleaved at the extracellular domain to
release the soluble form of ACE into the extracellular
fluid, including the blood [29-31]. ACE shedding is nega-
tively regulated by the cytoplasmic domain of ACE, a
domain that is not required for recognition by the ACE-
secretase. Shedding of ACE is regulated by calmodulin
(CaM), which binds to the cytoplasmic domain of ACE
[32]. Dissociation of CaM from the cytoplasmic domain
of ACE stimulates the cleavage secretion.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
was originally considered to be a glycolytic protein
involved in energy generation. Recent results have shown,
however, that it is a multifunctional protein with
cytosolic, nuclear and perinuclear localizations [33]
(Table 1). Briefly, GAPDH becomes nitrosylated in the
presence of high NO concentrations in the cytosol. After
its nitrosylation, the protein interaction of GAPDH and
Siah1 is significantly augmented. The GAPDH-Siah1 com-
plex then is translocated to the nucleus. Siah1 is a compo-
nent of a multiprotein E3 ubiquitin ligase complex that
targets nuclear proteins for destruction via the proteas-
ome. The GAPDH-Siah1 complex in the nucleus has been
shown to facilitate the degradation of nuclear substrates,
which results in cell death [34]. Figure 4 summarizes the
role of GAPDH in apoptosis. GAPDH is a further repre-
sentative example for the importance of the structure-
function relationship of protein species. This example,
among others, demonstrates the urgent need for a system-
atic nomenclature taking the structure-function relation-
ship into account.
As a consequence of the new paradigm of the gene-protein
relationship, we propose that the identity of each protein
should be described more precisely in the future. Already
in 1996 Jungblut et al. introduced and defined [1] the
term protein species for proteins which are derived from a
single gene but differ in protein chain length and/or post-
translational modification. The term "protein species" [2],
in this sense, is central to the new paradigm and the appli-
Protein species (sACE P12821; g-ACE P22966) derived from the angiotensin-converting enzyme gene
Figure 3
Protein species (sACE P12821; g-ACE P22966) derived from the angiotensin-converting enzyme gene. CaM:
calmodulin.
DNA
sACE P21821
1-1306
sACE P21821
30-1232
Ribosome
sACE P21821
30-1306
RNA
m-RNA A
m-RNA B
gACE P22966
1-732
sACE P21821
30-1306
CaM
- CaM
gACE P22966
32-732
CaM
gACE P22966
32-684
regulation of
blood pressure
fluid homeostasis
electrolyte balance
regulation of male fertility
gACE P22966
32-732
- CaM
+ CaM
+ CaM

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cation of the protein species concept enables the develop-
ment of a systematic nomenclature required for a
comprehensive understanding of different functions of a
protein.
Protein species nomenclature based on the protein species
concept
For a protein species nomenclature we need to distinguish
various levels: At the gene level a gene may have one or
more than one transcript (transcript level); each transcript
may lead to one or more than one translation. For the pro-
tein species nomenclature the following levels must be
considered:
1. The initial protein species is the primary translation
product. One would expect that, usually, one distinct
transcript would lead to one distinct primary transla-
tion product. However, some of these initial protein
species may be indistinguishable although derived
from different transcripts. In Caenorhabditis elegans, for
example, there are four genes coding for actins. The
protein sequences encoded by genes 1 and 3 are iden-
tical (UniProt Knowledgebase AC P10983) [35]. On
Protein species (GAPDH P04406) derived from the GAPDH gene
Figure 4
Protein species (GAPDH P04406) derived from the GAPDH gene. Siah1: protein interacting with GAPDH.
DNA
GAPDH
Ribosome
RNA
NO - Stress
m-RNA A
NO
GAPDH
NAD
GAPDH
NO
NAD
Siah1
Q8IUQ4
GAPDH
NO
NAD
Cell death
Siah1
Q8IUQ4
Table 1: Some examples of different functions and localizations of GAPDH.
Function/Cellular roleLocalization PTM*Interaction partnerReference
Apoptosis (Figure 4)
Plasma membrane fusion
Microtubule structure and function
Translational regulation
Cell transport properties
Transcription control
Nucleus
Membrane
Cytoplasm
-
Secretory vesicles
Nucleus
Nitrosylation
-
-
-
Phosphorylation
-
Siah
Anion exchanger protein
Tubulin
5V and 3VUTR mRNA sequence
PKCL/E
Oct-1 transcription factor +
promyelocytic leukemia
nuclear bodies
Telomeres
tRNA precursors
Ap4A
-
[36]
[37]
[38]
[39]
[40]
[41]
[42]
Nucleus
Nucleus
-
Nuclear envelope
-
-
-
-
[43]
[44]
[45,46]
[47]
Intranuclear transport
Cell proliferation
Nuclear membrane
fusion
*post-translational modification

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the other hand, some unique transcripts may lead -
due to different transcription start sites - to different
initial protein species, and there are even a few cases
where a human gene gives rise to an mRNA that seems
to be bistronic and encodes for two different products.
Examples are TREX2 (UniProt Knowledgebase AC
Q9BQ50) [35] and UCHL5IP (UniProt Knowledge-
base AC Q99871) [35]. Most mRNAs that encode
UCHL5IP also include the N-terminal part of TREX2.
The initial protein species may be the final functional
protein species, or may undergo further processing
such as proteolytic cleavage and/or chemical modifi-
cation.
2. Proteolytically processed protein species. The pro-
tein processing procedure may lead to the final func-
tional protein species or the protein species may still
be subject to additional chemical modification.
3. A protein may undergo additional chemical modifi-
cation such as acetylation, phosphorylation, etc.
On a species scale, on all these levels, we also have to con-
sider nucleotide polymorphisms (SNPs, insertions, dele-
tions, etc) that may change the amino acid composition of
the derived protein species. Because there is currently no
systematic nomenclature available for the exact descrip-
tion of protein species, we present such a nomenclature.
The following combination of terms is suggested for a
complete description of an individual protein species.
Each describing parameter is contained in square brackets.
An overview of the individual terms of the protein species
nomenclature is given in Table 2. Below the detailed
description of the terms is given.
Gene level
The name of a protein species starts with a descriptor
which is identical with its entry name in the knowledge-
base UniProt [35], containing the term for the gene name
and the term for the species. E.g., the descriptor for the
human gene coding an angiotensin-converting-enzyme is
G_ACE_human. If no analytical data are available the
descriptor for the gene gives far more reliable information
about the identity of the protein species for the descrip-
tion of biological experiments than synonyms which are
still in wide use.
Nucleotide polymorphism level
Describe the exact protein isoform adding the accession
number of the record describing the polymorphism. E.g.,
SNP_rs10853044 describes an SNP of the human angi-
otensin-converting-enzyme which is responsible for the
replacement of Leu by Pro at the amino acid position 132
of P12821-1_1.
Initial Protein Species level
The identity of a protein species giving access to its initial
amino acid sequence, which is observed directly after its
protein synthesis (initial protein species level), is defined
by the protein database accession number and sequence
version number. In the nomenclature suggested here, the
name of a protein starts with AC_ followed by the acces-
sion number and the sequence version number. For exam-
ple, the name for the somatic species of the human
angiotensin-converting enzyme is [AC_ P12821-1_1]. In
many cases accession numbers for different splicing vari-
ants are already available. For example, the two splicing
variants of the angiotensin-converting enzyme are desig-
nated P12821 (representing the somatic species) and
P22966 (representing the testis species).
The human serine/threonine-protein phosphatase also
exists in several splicing variants. Here the name of the
regulatory subunit beta species is [AC_ Q15173_2]. The
name for the species beta-2 is [AC_ Q15173-2_2]. The
splicing variant is indicated here by -2. The sequence ver-
sion number is designated by _2.
If no accession number exists for the exact transcript trans-
lation (in the case of an alternative spliced protein species
without a splice isoform number), describe the exact tran-
script translation starting with S_ (S for splicing). S should
be followed by D for deletion (SD_), if amino acids are
missing compared to the canonical protein record
described by the protein accession and sequence version
number. If, say, the unspliced protein species consists of
100 amino acids and the spliced species is missing the
amino acids 70-79 the term is [SD_70-79].
If a peptide chain is inserted as compared to the canonical
protein record, the term starts with SI_ followed by the
number of the amino acid preceding the inserted peptide
chain. This number is followed by the amino acid
sequence written in the one-letter code. E.g., if the peptide
chain LLELFVMFL is inserted at the position of amino acid
number 43 the term then is [SI_43_ LLELFVMFL].
An exchange (E) of one amino acid or several amino acids
can be designated [SE_38_RQELWQG_SKEHWNQ].
Where 38 indicates the number of the first exchanged
amino acid of the peptide. SKEHWNQ is the peptide
present in the protein species, which was substituted for
RQELWQG.
Proteolytically processed protein species level
Describe the protein sequence after proteolytic process-
ing, starting with T_: e.g., [T_1-17] indicates that the first
17 amino acids were removed.

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Chemically modified protein level
Describe the post-translational modification(s), starting
with P_ followed by the number of the amino acid that
has been modified and the UniMod accession number
[6]. As an example take: [P_33_21]. Here 33 indicates the
amino acid that is phosphorylated and 21 is the accession
number for phosphorylation from UniMod.
Cofactor level
Describe the identity of non-covalently bound cofactors
starting with C_.
Further descriptors [X,Y] can be added, if necessary. As an
example we give the combination of descriptors for the
full description of the human somatic angiotensin-con-
verting enzyme, including an SNP located in the mem-
brane of vascular smooth muscle cells:
[G_ACE_human]+ [SNP_rs10853044]+ [AC_P12821-
1_1]+ [T_1-29/1233-1306]+ [P_N-linked-carbohydrate
chains at 38, 54, 74, 111, 146, 160, 318, 445, 509, 677,
695, 714, 760, 942, 1191)+[P_21]+ [C_Zn(2x)].
The data for the carbohydrate chains were taken from the
UniProt Knowledgebase [35].
This nomenclature takes the structure-function relation-
ship into account. Furthermore, it is well suited for data-
base searches and will provide a more reliable foundation
for systems biology approaches. We recommend that in
future publications on protein species authors use this
nomenclature, presenting the complete protein species
term at least in the Abstract and in the Introduction of
their manuscript. Since the complete protein species
descriptor is long, the author should define a short form
substitute of the protein species for use in the rest of the
paper.
If not every detail of the exact chemical composition can
be determined, the author should include those terms
which are accessible. As a minimum, the entry number
according to UniProt Knowledgebase [35] should be
given.
Protein databases such as the UniProt Knowledgebase
[35] will ideally create unique identifiers for each protein
species identified. This will take time to implement, how-
ever, and even then the assignment of such unique identi-
fiers will lag behind the detection of new protein species.
The creation and use of protein species descriptors will
therefore be of permanent value - unless it in its turn is
replaced someday by an improved nomenclature.
Conclusion
We have presented a scheme for the accurate and detailed
description of protein species. We propose that future
investigations of protein function focusing on defined
protein species use this protein species terminology which
includes the complete terms necessary for the comprehen-
sive description of a protein species. It is desirable not
only to characterize the protein function, but to identify
the target protein species in question with a sequence cov-
erage as high as possible - ideally 100% - and the identifi-
cation of all post-translational modifications. These
requirements should also be addressed in proteome ana-
lytical investigations, by digesting protein mixtures not
Table 2: Overview of the individual terms (descriptors) of the protein species nomenclature
LevelComment Descriptor
A: Gene
B: Nucleotide polymorphism
C: Initial amino acid sequence of the protein
synthesized at the ribosome
Entry name (gene name + species)
SNP Accession number
Accession number (AC) + sequence version (_1)
G_ACE_humana
SNP_rs10853044
AC_ P12821-1_1
Splicing variant (S) with deletion _ localisation of the
deletion
Splicing variant with insertion _ localisation of the
insertion
Splicing variant with an exchange of amino acids
Truncated amino acids: 1-17: the first 17 amino acids
were removed
Position of the modified amino acid
Identity of non-covalently bound cofactors
Can be added, if necessary
SD_70-79
SI_43_ LLELFVMFL
SE_38_RQELWQG_SKEHWNQb
T_1-17D: Proteolysis
E: Posttranslational Modification (P)
F: Cofactor (C)
G: Further descriptors
P_33_21c
C_Zn(2x)d
X, Y
aaccording to the term in the UniProt knowledgebase [35]
b38 indicates the number of the first exchanged amino acid of the peptide. SKEHWNQ is the peptide present in the protein species, which was
substituted for RQELWQG.
c the first number (here: 33) gives the position of the modified amino acid; the second number gives the type of the post-translational modification
according to the UniMod [6] accession number (here: 21 indicating a phosphorylation)
d here the term indicates the presence of 2 cations of Zn

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only with trypsin but by several other proteases in paral-
lel, thus achieving higher sequence coverage. In the short
term in many cases it may not be possible to achieve
100% sequence coverage and/or the identification of
every PTM of individual protein species. Nonetheless,
even in these cases use of the protein species nomencla-
ture will be helpful, indicating the current level of knowl-
edge of the exact chemical structure and giving hints as to
the direction further investigations should take.
In summary, we propose a tool for the storage of informa-
tion on protein species which makes accessible a clear
chemical description of protein molecules.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
HS developed the nomenclature, wrote the text, and con-
tributed Figures 2 to 4. Parts of the text and Figure 1 were
prepared by PRJ, who contributed - together with HGH
and RA - to the concept of the nomenclature and critically
revised the text.
Acknowledgements
Anna Walduck and Peter Germain are acknowledged for editorial support.
References
1.Jungblut P, Thiede B, Zimny-Arndt U, Muller EC, Scheler C, Witt-
mann-Liebold B, Otto A: Resolution power of two-dimensional
electrophoresis and identification of proteins from gels. Elec-
trophoresis 1996, 17:839-47.
2.Jungblut PR, Holzhütter HG, Apweiler R, Schlüter H: The Specia-
tion of the Proteome. Chem Cent J 2008, 2:16.
3.Blanchard JM, Brissac C, Jeanteur P: Characterization of a protein
species isolated from HeLa cell cytoplasm by affinity chro-
matography on polyadenylate-sepharose. Proc Natl Acad Sci
USA 1974, 71:1882-6.
4. O'Farrell PH: High resolution two-dimensional electrophore-
sis of proteins. J Biol Chem 1975, 250:4007-21.
5. Joint Commission on Biochemical Nomenclature IUPAC-IUBMB:
Nomenclature of multiple forms of enzymes. In Biochemical
Nomenclature and Related Documents 2nd edition. Edited by: Liébecq
C. Colchester: Portland Press; 1992.
6.UNIMOD - Protein modifications for mass spectrometry: [http://
www.unimod.org/modifications_list.php].
7.Creasy DM, Cottrell JS: Protein modifications for mass spec-
trometry. Proteomics 2004, 4:1534-6.
8.Nielsen ML, Savitski MM, Zubarev RA: Extent of modifications in
human proteome samples and their effect on dynamic range
of analysis in shotgun proteomics. Mol Cell Proteomics 2006,
5:2384-91.
9.Sun SC: Deubiquitylation and regulation of the immune
response. Nat Rev Immunol 2008, 8:501-11.
10.Klose J, Kobalz U: Two-dimensional electrophoresis of pro-
teins: an updated protocol and implications for a functional
analysis of the genome. Electrophoresis 1995, 16:1034-59.
11.Jungblut P, Thiede B: Protein identification from 2-DE gels by
MALDI mass spectrometry. Mass Spectrom Rev 1997, 16:145-62.
12.McCormack AL, Schieltz DM, Goode B, Yang S, Barnes G, Drubin D,
Yates JR 3rd: Direct analysis and identification of proteins in
mixtures by LC/MS/MS and database searching at the low-
femtomole level. Anal Chem 1997, 69:767-76.
13.Kelleher N: Top-down proteomics. Anal Chem 2004,
76:197A-203A.
14.Han X, Jin M, Breuker K, McLafferty FW: Extending top-down
mass spectrometry to proteins with masses greater than 200
kilodaltons. Science 2006, 314(5796):109-12.
Roth M, Forbes A, Boyne M, Kim Y, Robinson D, Kelleher N: Precise
and parallel characterization of coding polymorphisms,
alternative splicing, and modifications in human proteins by
mass spectrometry. Mol Cell Proteomics 2005, 4:1002-8.
Schmidt F, Donahoe S, Hagens K, Mattow J, Schaible U, Kaufmann S,
Aebersold R, Jungblut PR: Complementary analysis of the Myco-
bacterium tuberculosis proteome by two-dimensional elec-
trophoresis and isotope-coded affinity tag technology. Mol
Cell Proteomics 2004, 3(1):24-42.
Wu S, Lourette NM, Tolic N, Zhao R, Robinson EW, Tolmachev AV,
Smith RD, Pasa-Tolic L: An integrated top-down and bottom-up
strategy for broadly characterizing protein isoforms and
modifications. J Proteome Res 2009, 8(3):1347-57.
Scheler C, Muller E, Stahl J, Muller-Werdan U, Salnikow J, Jungblut P:
Identification and characterization of heat shock protein 27
protein species in human myocardial two-dimensional elec-
trophoresis patterns. Electrophoresis 1997, 18:2823-31.
Klose J, Nock C, Herrmann M, Stuhler K, Marcus K, Bluggel M, Krause
E, Schalkwyk LC, Rastan S, Brown SD, Bussow K, Himmelbauer H,
Lehrach H: Genetic analysis of the mouse brain proteome. Nat
Genet 2002, 30(4):385-93.
Mattow J, Jungblut PR, Muller E, Kaufmann S: Identification of
acidic, low molecular mass proteins of Mycobacterium
tuberculosis strain H37Rv by matrix-assisted laser desorp-
tion/ionization and electrospray ionization mass spectrome-
try. Proteomics 2001, 1:494-507.
Strahl B, Allis C: The language of covalent histone modifica-
tions. Nature 2000, 403:41-5.
Brumbaugh J, Phanstiel D, Coon JJ: Unraveling the histone's
potential: a proteomics perspective. Epigenetics 2008, 3:254-7.
Sims RJ 3rd, Reinberg D: Is there a code embedded in proteins
that is based on post-translational modifications? Nat Rev Mol
Cell Biol 2008, 9:815-20.
Mayer MP, Bukau B: Hsp70 chaperones: cellular functions and
molecular mechanism. Cell Mol Life Sci 2005, 62(6):670-84.
Morishima N: Control of cell fate by Hsp70: more than an eva-
nescent meeting. J Biochem (Tokyo) 2005, 137(4):449-53.
Okkels L, Muller E, Schmid M, Rosenkrands I, Kaufmann S, Andersen
P, Jungblut PR: CFP10 discriminates between nonacetylated
and acetylated ESAT-6 of Mycobacterium tuberculosis by
differential interaction. Proteomics 2004, 4:2954-60.
Warren EH, Vigneron NJ, Gavin MA, Coulie PG, Stroobant V, Dalet
A, Tykodi SS, Xuereb SM, Mito JK, Riddell SR, Eynde BJ Van den: An
antigen produced by splicing of noncontiguous peptides in
the reverse order. Science 2006, 313:1444-7.
Kumar RS, Thekkumkara TJ, Sen GC: The mRNAs encoding the
two angiotensin-converting isozymes are transcribed from
the same gene by a tissue-specific choice of alternative tran-
scription initiation sites. J Biol Chem 1991, 266:3854-62.
29.Ramchandran R, Sen I: Cleavage processing of angiotensin-con-
verting enzyme by a membrane-associated metallopro-
tease. Biochemistry 1995, 34:12645-52.
30.Ramchandran R, Kasturi S, Douglas JG, Sen I: Metalloprotease-medi-
ated cleavage secretion of pulmonary ACE by vascular
endothelial and kidney epithelial cells. Am J Physiol 1996, 271(2
Pt 2):H744-51.
31.Sadhukhan R, Santhamma KR, Reddy P, Peschon JJ, Black RA, Sen I:
Unaltered cleavage and secretion of angiotensin-converting
enzyme in tumor necrosis factor-alpha-converting enzyme-
deficient mice. J Biol Chem 1999, 274:10511-6.
32.Chattopadhyay S, Santhamma KR, Sengupta S, McCue B, Kinter M,
Sen GC, Sen I: Calmodulin binds to the cytoplasmic domain of
angiotensin-converting enzyme and regulates its phosphor-
ylation and cleavage secretion. J Biol Chem 2005,
280(40):33847-55.
33.Sirover MA: New insights into an old protein: the functional
diversity of mammalian glyceraldehyde-3-phosphate dehy-
drogenase. Biochim Biophys Acta 1999, 1432:159-184.
34.Hara MR, Snyder SH: Nitric Oxide-GAPDH-Siah: A Novel Cell
Death Cascade. Cell Mol Neurobiol 2006, 26:527-38.
35.The UniProt Consortium: The Universal Protein Resource
(UniProt). Nucleic Acids Res 2009, 37:D169-D174.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.

Page 10

Chemistry Central Journal 2009, 3:11 http://journal.chemistrycentral.com/content/3/1/11
Page 10 of 10
(page number not for citation purposes)
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36.Hara MR, Cascio MB, Sawa A: GAPDH as a sensor of NO stress.
Biochim Biophys Acta 2006, 1762:502-509.
Hessler RJ, Blackwood RA, Brock TG, Francis JW, Harsh DM, Smolen
JE: Identification of glyceraldehyde-3-phosphate dehydroge-
nase as a Ca2+-dependent fusogen in human neutrophil
cytosol. J Leukoc Biol 1998, 63(3):331-6.
Muronetz VI, Wang ZX, Keith TJ, Knull HR, Srivastava DK: Binding
constants and stoichiometries of glyceraldehyde 3-phos-
phate dehydrogenase-tubulin complexes. Arch Biochem Biophys
1994, 313:253-260.
Schultz DE, Hardin CC, Lemon SM: Specific interaction of glycer-
aldehyde-3-phosphate dehydrogenase with the 5V-nontrans-
lated RNA of hepatitis A virus. J Biol Chem 1996,
271:14134-14142.
Tisdale EJ: Glyceraldehyde-3-phosphate dehydrogenase is
required for vesicular transport in the early secretory path-
way. J Biol Chem 2001, 276:2480-2486.
Zheng L, Roeder RG, Luo Y: S phase activation of the histone
H2B promoter by OCA-S, a coactivator complex that con-
tains GAPDH as a key component. Cell Mol Life Sci 2003,
114:255-266.
Carlile GW, Tatton WG, Borden DLB: Demonstration of a RNA-
dependent nuclear interaction between the promyelocytic
leukaemia protein and glyceraldehyde-3-phosphate dehy-
drogenase. Biochem J 1998, 335:691-696.
Sundararaj KP, Wood RE, Ponnusamy S, Salas AM, Szulc Z, Bielawska
A, Obeid LM, Hannun YA, Ogretmen B: Rapid shortening of tel-
omere length in response to ceramide involves the inhibition
of telomere binding activity of nuclear glyceraldehyde-3-
phosphate dehydrogenase. J Biol Chem 2004, 279:6152-6162.
Singh R, Green MR: Sequence-specificity binding of transfer
RNA by glyceraldehyde-3-phosphate dehydrogenase. Science
1993, 259:365-368.
Baxi MD, Vishwanatha JK: Uracil DNA lycosylase/glyceralde-
hyde-3-phosphate dehydrogenase is an Ap4A binding pro-
tein. Biochemistry 1995, 34:9700-9707.
Xing C, LaPorte JR, Barbay JK, Myers AG: Identification of
GAPDH as a protein target of the saframycin antiprolifera-
tive agents. Proc Natl Acad Sci USA 2004, 101:5862-5866.
Nakagawa T, Hirano Y, Inomata A, Yokota S, Miyachi K, Kaneda M,
Umeda M, Furukawa K, Omata S, Hoarigome T: Participation of a
fusogenic protein, glyceraldehyde-3-phosphate dehydroge-
nase, in nuclear membrane assembly. J Biol Chem 2003,
278:20395-20404.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.