High sequence coverage of proteins isolated from liquid separations of breast cancer cells using capillary electrophoresis-time-of-flight MS and MALDI-TOF MS mapping.
ABSTRACT A method has been developed for high sequence coverage analysis of proteins isolated from breast cancer cell lines. Intact proteins are isolated using multidimensional liquid-phase separations that permit the collection of individual protein fractions. Protein digests are then analyzed by both matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) peptide mass fingerprinting and by capillary electrophoresis-electrospray ionization (CE-ESI)-TOF MS peptide mapping. These methods can be readily interfaced to the relatively clean proteins resulting from liquid-phase fractionation of cell lysates with little sample preparation. Using combined sequence information provided by both mapping methods, 100% sequence coverage is often obtained for smaller proteins, while for larger proteins up to 75 kDa, over 90% coverage can be obtained. Furthermore, an accurate intact protein MW value (within 150 ppm) can be obtained from ESI-TOF MS. The intact MW together with high coverage sequence information provides accurate identification. More notably the high sequence coverage of CE-ESI-TOF MS together with the MS/MS information provided by the ion trap/reTOF MS elucidates posttranslational modifications, sequence changes, truncations, and isoforms that may otherwise go undetected when standard MALDI-MS peptide fingerprinting is used. This capability is critical in the analysis of human cancer cells where large numbers of expressed proteins are modified, and these modifications may play an important role in the cancer process.
- [show abstract] [hide abstract]
ABSTRACT: This review intends not only to discuss the current possibilities to gain 100% sequence coverage for proteins, but also to point out the critical limits to such an attempt. The aim of 100% sequence coverage, as the review title already implies, seems to be rather surreal if the complexity and dynamic range of a proteome is taken into consideration. Nevertheless, established bottom-up shotgun approaches are able to roughly identify a complete proteome as exemplary shown by yeast. However, this proceeding ignores more or less the fact that a protein is present as various protein species. The unambiguous identification of protein species requires 100% sequence coverage. Furthermore, the separation of the proteome must be performed on the protein species and not on the peptide level. Therefore, top-down is a good strategy for protein species analysis. Classical 2D-electrophoresis followed by an enzymatic or chemical cleavage, which is a combination of top-down and bottom-up, is another interesting approach. Moreover, the review summarizes further top-down and bottom-up combinations and to which extent middle-down improves the identification of protein species. The attention is also focused on cleavage strategies other than trypsin, as 100% sequence coverage in bottom-up experiments is only obtainable with a combination of cleavage reagents.Amino Acids 07/2011; 41(2):291-310. · 3.91 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: In-gel digestion of gel-separated proteins is a major route to assist in proteomics-based biological discovery, which, however, is often embarrassed by its inherent limitations such as the low digestion efficiency and the low recovery of proteolytic peptides. For overcoming these limitations, many efforts have been directed at developing alternative methods to avoid the in-digestion. Here, we present a new method for efficient protein digestion and tryptic peptide recovery, which involved electroblotting gel-separated proteins onto a PVDF membrane, excising the PVDF bands containing protein of interest, and dissolving the bands with pure DMF (> or =99.8%). Before tryptic digestion, NH(4)HCO(3) buffer was added to moderately adjust the DMF concentration (to 40%) in order for trypsin to exert its activity. Experimental results using protein standards showed that, due to actions of DMF in dissolving PVDF membrane and the membrane-bound substances, the proteins were virtually in-solution digested in DMF-containing buffer. This protocol allowed more efficient digestion and peptide recovery, thereby increasing the sequence coverage and the confidence of protein identification. The comparative study using rat hippocampal membrane-enriched sample showed that the method was superior to the reported on-membrane tryptic digestion for further protein identification, including low abundant and/or highly hydrophobic membrane proteins.Electrophoresis 09/2009; 30(20):3626-35. · 3.26 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: As a complementary approach to 2D-PAGE, multidimensional liquid chromatography (MDLC) separation methods have been widely applied in all kinds of biological sample investigations. MDLC coupled with mass spectrometry is playing an important role in proteome research owing to its high speed, high resolution and high sensitivity. Among MDLC strategies, ion-exchange chromatography together with reversed-phase LC is still a most widely used chromatography in proteome analysis; other chromatographic methods are also frequently used in protein prefractionations. Recent MDLC technologies and applications to a variety of proteome analyses have achieved great development. The diversity of combinations of different chromatography modes to set up MDLC systems was demonstrated and discussed. Novel developments of MDLC techniques such as ultra-pressure system, array-based separation and monolithic material are also included in this article.Expert Review of Proteomics 10/2010; 7(5):665-78. · 3.90 Impact Factor
High S equenc e Coverage of Proteins Isolated from
Liquid S eparations of Breast Canc er Cells Using
Capillary Elec trophoresis-T ime-of-Flight MS and
MALDI-T OF MS Mapping
K an Zhu,²J eongkwon K im,² ,³Chul Y oo,²Fred R. Miller,§and David M. Lubman*,²
Department of Chemistry, The University of Michigan, Ann Arbor, Michigan 48109-1055, and
Barbara Ann Karmanos Cancer Institute, Wayne State University, Detroit, Michigan 48201
A method has been developed for high sequence coverage
analysis of proteins isolated from breast cancer cell lines.
Intact proteins are isolated using multidimensional liquid-
phase separations that permit the collection of individual
protein fractions. Protein digests are then analyzed by
both matrix-assisted laser desorption/ionization time-of-
flight mass spectrometry (MALDI-TOF MS) peptide mass
fingerprinting and by capillary electrophoresis-electro-
spray ionization (CE-ESI)-TOF MS peptide mapping.
These methods can be readily interfaced to the relatively
clean proteins resulting from liquid-phase fractionation
of cell lysates with little sample preparation. Using com-
bined sequence information provided by both mapping
methods, 100% sequence coverage is often obtained for
smaller proteins, while for larger proteins up to 75 kDa,
over 90% coverage can be obtained. Furthermore, an
accurate intact protein MW value (within 150 ppm) can
be obtained from ESI-TOF MS. The intact MW together
with high coverage sequence information provides ac-
curate identification. More notably the high sequence
coverage of CE-ESI-TOF MS together with the MS/MS
information provided by the ion trap/reTOF MS elucidates
posttranslational modifications, sequence changes, trun-
cations, and isoforms that may otherwise go undetected
when standard MALDI-MS peptide fingerprinting is used.
This capability is critical in the analysis of human cancer
cells where large numbers of expressed proteins are
modified, and these modifications may play an important
role in the cancer process.
Proteomic techniques have become important in the study of
disease by providing an integrated view of disease at the protein
level. The identification of protein markers may be used for
diagnosis and prognosis of disease1-3as well as targets for
development of new drugs.4,5The development of procedures for
separating proteins from complex mixtures and for generating
structural information from the proteins of interest are two key
areas in proteome research. One such method for studying
complex protein mixtures involves using 2-D-PAGE to separate
large numbers of proteins from cell lysates where protein
structural information is obtained by analyzing enzymatic digests
of gel spots with matrix-assisted laser desorption/ionization time-
of-flight mass spectrometry (MALDI-TOF MS).6-9Proteins sepa-
rated by gels are not directly compatible with MALDI analysis. A
number of procedures including spot excision, destaining, enzy-
matic digestion, extraction of peptides into solution, and spotting
the sample are required prior to MALDI-TOF analysis.10A major
drawback of the method is that only a limited coverage of the
protein sequence is obtained from MALDI-TOF MS due to ion
suppression and varying ionization efficiencies for different pep-
tides.11The result is that in complex proteomes such a peptide
map may provide incorrect identifications, depending upon the
parameters with which the database is searched. Furthermore,
the limited coverage often does not identify the presence of
posttranslational modifications, which are critical to protein
function and dysfunction.12,13
The development of methods to analyze proteins with high
sequence coverage is essential in the field of proteomics. High
* Corresponding author: (tel) 734-764-1669, (fax) 734-615-8108, (e-mail)
²The University of Michigan.
³Present address: Environmental Molecular Sciences Laboratory, Pacific
Northwest National Laboratory, P.O. Box 999, Richland, WA 99352.
§Wayne State University.
(1) Lawrie, L. C.; Fothergill, J. E.; Murray, G. I. Lancet Oncol. 2001, 2, 270-
(2) Srinivas, P. R.; Verma, M.; Zhao, Y.; Srivastava, S. Clin. Chem. 2002, 48
(3) Petricoin, E. F.; Zoon, K. C.; Kohn, E. C.; Barrett, J. C.; Liotta, L. A. Nat.
Rev. Drug Discovery 2002, 1, 683-695.
(4) Figeys, D. Anal. Chem. 2002, 413A-419A.
(5) Vercoutter-Edouart, A. S.; Lemoine, J.; Le Bourhis, X.; Louis, H.; Boilly, B.;
Nurcombe, V.; Revillion, F.; Peyrat, J. P.; Hondermarck, H. Cancer Res.
2001, 61 (1): 76-80.
(6) Henzel, J. W.; Billeci, T. M.; Stults, J. T.; Wong, S. C.; Grimely, C.; Watanabe,
C. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 5011-5015.
(7) Yates, J. R. J. Mass Spectrom. 1998, 33, 1-9.
(8) Liang, X.; Bai, J.; Liu, Y. H.; Lubman, D. M. Anal. Chem. 1996, 68, 1012-
(9) Loo, R. R. O.; Stevenson, T. I.; Mitchell, C.; Loo, J. A.; Andrews, P. C. Anal.
Chem. 1996, 68, 1910-1917.
(10) Lahm, H. W.; Langen, H. Electrophoresis 2000, 21, 2105-2114.
(11) Krause, E.; Wenschuh, H.; Jungblut, P. R. Anal. Chem. 1999, 71, 4160-
(12) Minamoto, T.; Buschmann, T.; Habelhah, H.; Matusevich, E.; Tahara, H.;
Boerresen-Dale, A. L.; Harris, C.; Sidransky, D.; Ronai, Z. Oncogene2001,
(13) Wilkins, M. R.; Gasteiger, E.; Gooley, A. A.; Herbert, B. R.; Molloy, M. P.;
Binz, P. A.; Ou, Keli; Sanchez, J. C.; Bairoch, A.; Williams, K. L.;
Hochstrasser, D. F. J. Mol. Biol. 1999, 289, 645-657.
Anal. Chem. 2003, 75, 6209-6217
10.1021/ac0346454 CCC: $25.00
Published on Web 10/02/2003
© 2003 American Chemical Society
Analytical Chemistry, Vol. 75, No. 22, November 15, 2003
sequence coverage eliminates false identifications in database-
searching algorithms and provides a means for identifying the
presence of posttranslational modifications (PTMs). A number of
methods havebeendevelopedtoachievehigh sequencecoverage
and to eliminate the use of 2-D gel technology. In recent work by
Yates and co-workers, variant hemoglobins obtained from blood
were digested with three different enzymes and analysis was
performed on the combined enzymatic peptide mixtures with
microcapillary liquid chromatography (LC)-MS/MS.14Using the
combination of three enzymes, asequence coverage of >99%was
obtained. An improvement on the method is achieved by com-
bining three proteolytic peptide mixtures followed by MUDPIT
analysis.15However, lack of intact molecular weight information
prevents it from predicting PTMs and making decisions if the
modification is not detected. In other work, using complementary
results from ESI and MALDI, Prokai et al. obtained 95+%
sequence coverage for cytolysin proteins purified from sea
anemone Stichodactyla helianthus.16In all three cases, the use of
high sequence coverage revealed protein variants and modifica-
tions. Smith et al. also achieved 100% sequence coverage for
transform ion cyclotron resonance.17Another alternative method
used by Kelleher and co-workers has been the ESI-based ªtop-
downº approach, which has achieved 100%sequence coverage.18
However, this method has thus far been applied to relatively
simple organisms and for proteins under 40 kDa.
In this work, amethod is introduced toachieve high sequence
coverage of proteins isolated from real cellular samples. A liquid-
based 2-D fractionation of proteins from cell lysates produces
purified proteins in the liquid phase. The method has been
introduced in prior work to map cellular proteins as a means to
search for markers of disease.19-21Greater than 50%of human
proteins were found to be modified; thus, many proteins in the
humanlysates weredifficulttoidentify with confidencebasedupon
the tryptic peptide map alone.22A major advantage of the 2-D
methodis theproductionofsubstantial amounts ofhighly purified
proteins isolated in the liquid phase. As a result, proteins are
digestedanddirectly analyzedby acombinationofMS techniques
(capillary electrophoresis-electrospray ionization (CE-ESI)-MS
andMALDI-TOF MS) with minimal samplepreparation. CE-ESI-
MS can provide high sequence coverage of protein digests often
approaching total coveragefor proteins under 20kDa.23,24MALDI-
TOF MS often provides improved detection for peptides that are
not detected by ESI-MS. The result is that often >90%coverage
can be obtained even for large proteins using a combination of
Inthepresentwork, theuseof2-D liquidseparations combined
with CE-ESI-MS andMALDI-TOF MS for high sequencecoverage
of selected proteins isolated from breast cancer cell lines is
demonstrated. Over a MW range of 4000-70 000, >90%of the
sequence can be obtained from a single tryptic digest. The
presenceofproteins intheliquidphaseallows directdetermination
of an accurate MW value that is used to determine the presence
of PTMs. The MW together with the high sequence coverage
afforded by this method and the use ofMS/MS provides ameans
to determine the identification and sites of PTMs as well as the
presence of sequence deletions and additions and resulting
isoforms. Different isoforms of lamin A, a truncation of HSP60,
and acetylation of thymosin ?4 were distinguished using the
method. Theseprocedures providestructural informationessential
in studies of cancer progression and biomarker identification.
EX PERIMENTAL SECTION
MCF10Ca1DCL1 Cells and Lysis. MCF10Ca1d clone 1
(CA1d) is afully malignant humanbreast cancer line.25Cells were
grown in monolayer on plastic in DMEM/F12 medium supple-
mentedwith 5%horseserum, 10mM N-2-hydroxyethylpiperazine-
N′-2-ethanesulfonic acid (HEPES). Adherent cells were harvested
in log phase (∼75-80%confluence). The growth medium was
aspirated, andthecells weregently washedwith sterilePBS buffer,
then scraped with arubber policeman, and stored in -80°C. The
cell pellets were lysed by adding 1.5mL of lysis buffer containing
8M urea, 2M thiourea, 0.5%(w/v) n-octyl ?-D-galactopyranoside,
50mM dithiothreitol, 10mM phenylmethanesulfonyl fluoride, and
10% (v/v)glycerol, vortexed for 2 min, and then left at room
temperature for 1 h. After the sample was lysed, the resulting
mixturewas centrifugedat15000rpmfor 20min. Thesupernatant
was collected, and Bradford assays (Bio-Rad, Hercules, CA) were
performed to quantify the amount of protein in the lysate. The
supernatant was removed, diluted to 18 mL with the isoelectric
focusing (IEF) running buffer, and introduced into the mini-
Rotofor (Bio-Rad, Hercules, CA) separation chamber for fraction-
ation. All chemicals were obtained from Sigma Chemical Co. (St.
Louis, MO) unless specified otherwise.
Liquid-Phase Isoelectric Focusing. The Bio-Rad Mini-
Rotofor was used to separate the cell extract by IEF in the first
dimension. Cell extract was mixed with IEF running buffer
composed of 8 M urea, 2 M thiourea, and 2%pH 3-10 Biolyte
(Bio-Rad). The Rotofor cell was loaded with 18mL of the mixture
and the separation controlled at a constant 12 W for 3.5 h.
Separated pI fractions were harvested into20tubes and stored in
-80°C until further analysis. pH measurements aretakenfor each
fractionusing anOrionpH meter (model 250A, Allometrics, Baton
Rouge, LA) and Accumet combination electrode (Fischer, Pitts-
Nonporous (NPS) Reversed-Phase Liquid Chromatogra-
phy. Briefly, NPS-RP HPLC separation is performed at aflow rate
(14) Gatlin, C. L.; Eng, J. K.; Cross, S. T.; Detter, J. C., Yates, J. R. III. Anal.
Chem. 2000, 72, 757-763.
(15) MacCoss, M. J.; McDonald, W. H.; Saraf, A.; Sadygov, R.; Clark, J. M.; Tasto,
J. J.; Gould, K. L.; Wolters, D.; Washburn, M.; Weiss, A.; Clark, J. I.; Yates,
J. R., III. Proc. Natl. Acad. Sci. U.S.A. 2002, 99 (12), 7900-7905.
(16) Stevens, S. M., Jr.; Kem, W. R.; Prokai, L. Rapid Commun. Mass Spectrom.
2002, 16, 2094-2101.
(17) Bruce, J. E.; Anderson, G. A.; Wen, J.; Harkewicz, R.; Smith, R. D. Anal.
Chem. 1999, 71, 2595-2599.
(18) Forbes, A. J.; Mazur, M. T.; Patel, H. M.; Walsh, C. T.; Kelleher, N. L.
Proteomics 2001, 1, 927-933.
(19) Wall, D. B.; Kachman, M. T.; Gong, S.; Hinderer, R.; Parus, S.; Misek, D.
E.; Hanash, S. M.; Lubman, D. M. Anal. Chem. 2000, 72, 1099-1111.
(20) Kachman, M. T.; Wang, H. X.; Schwartz, D. R.; Cho, K. R.; Lubman, D. M.
Anal. Chem. 2002, 74 (8), 1779-1791.
(21) Chong, B. E.; Hamler, R. L.; Lubman, D. M.; Ethier, S. P.; Rosenspire, A. J.;
Miller, F. R. Anal. Chem. 2001, 73 (6), 1219-1227.
(22) Wall, D. B.; Kachman, M. T.; Gong, S. Y.; Parus, S. J.; Long, M. W.; Lubman,
D. M. Rapid Commun. Mass Spectrom. 2001, 15 (18), 1649-1661.
(23) Jin, X. Y.; Kim, J.; Parus, S.; Lubman, D. M.; Zand, R. Anal. Chem. 1999,
(24) Cao, P.; Moini, M. Rapid Commun. Mass Spectrom. 1998, 12, 864-870.
(25) Santner, S. J.; Dawson, P. J.; Tait, L.; Soule, H. D.; Eliason, J.; Mohamed, A.
N.; Wolman, S. R.; Heppner, G. H.; Miller, F. R. Breast Cancer Res. Treat.
2002, 65, 101-110.
Analytical Chemistry, Vol. 75, No. 22, November 15, 2003
of 0.5 mL/min on an ODS IIIE (33 × 4.6 mm) column packed
with 1.5-µm C18 nonporous silica beads (Eprogen, Darien, IL).
About 300-350 µg of proteins from each Rotofor fraction was
loadedtotheNPS-RP columnfor optimumseparation. Thecolumn
is maintained at 65°C with aTimberline column heater (Boulder,
CO) in order to improve the resolution and speed of the
separation. Elution was performed using a water (A)/acetonitrile
(B) (0.1%(v/v) trifluoroacetic acid, TFA) gradient. The gradient
profile used was as follows: (1) 5-15%B in 1 min; (2) 15-25%B
in 2 min; (3) 25-31%B in 3 min; (4) 31-41%B in 10 min; (5)
41-47%B in 3 min; (6) 47-67%in 4 min; (7) 67-100%in 1 min;
(8) 100%B in2min; (9) 100-5%in1min. Theacetonitrileis 99.93%
HPLC grade(Sigma), andtheTFA was from1-mL sealedampules
(Sigma). The HPLC system was a Beckman model 127 HPLC
where the separation was monitored at 214 nm using a model
166detector. Theproteins separatedby NPS-HPLC werecollected
into 1.5-mL tubes using a Beckman SC-100 fraction collector
controlled by in-house software. Protein collection was performed
according tothe peaks detected fromthe HPLC separation where
45-50 fractions were obtained. The fractions typically contained
100-500 µL of liquid depending on the width of the peak.
NPS-RP-HPLC/ESI-TOF MS. Eluent from NPS-RP HPLC
was analyzed on-line using ESI-TOF MS (LCT, Micromass,
Manchester, U.K.) for intact MW analysis. The separation was
performed under the same conditions as in the previous section
except for the addition of 0.3%formic acid (Sigma) in both mobile
phases to improve ESI efficiency. In addition, in these experi-
ments, 40%of the eluent from the HPLC was split to the LCT.
The capillary voltage for electrospray was set at 3200 V, sample
cone at 40 V, extraction cone at 3 V, and reflection lens at 750 V.
Desolvation was accelerated by maintaining the desolvation
temperature at 300 °C and source temperature at 120 °C. The
nitrogengas flowwas controlledat650L/h. During theseparation,
onemass spectrumwas acquiredper second. Theintactmolecular
weight value was obtained by deconvoluting the combined ESI
spectra of the protein with MaxEnt 1 software (Micromass).
Protein Digestion. The proteins collected by NPS-RP-HPLC
separationweredried downto20µL using aSpeedVac (Labconco
Corp., Kansas City, MO), and 20 µL of 100 mM ammonium
bicarbonate was added to the solution before vortexing. A 0.5-µg
sample of L-1-tosylamido-2-phenylethyl chloromethyl ketone modi-
fied sequencing grade trypsin obtained fromPromega(Madison,
WI) was appliedfor tryptic digestion. Thedigestionwas performed
at 37 °C for 12 h. Enzymatic digestion was stopped by adding 1
µL of 10%(v/v) TFA, followed by completely drying the digested
samples with aSpeedVac. Prior toCE-MS analysis, peptides were
reconstituted in 1 µL of deionized water.
Capillary Electrophoresis Separation. The fused-silicacapil-
laries were purchased from Polymicro Technologies (Phoenix,
AZ). The fused-silica capillaries were internally coated according
to the procedure given by Bateman et al.26with minor modifica-
tions. A 50-cm-long fused-silica capillary with 110 µm o.d. × 40
µm i.d. was flushed with 1 M NaOH for 8 h using nitrogen gas
followed by rinsing with pure deionized water for 20 min.
Subsequently, thecapillary was flushedwith coating solution(10%
(w/v) Polybrene (Sigma), 3%(v/v) ethylene glycol (Sigma) in
water) for 10h. The capillary was then rinsed with pure deionized
water for 20 min. A buffer solution of pH 3 was prepared using
100 mM formic acid and ammonia. Electrokinetic injection was
used to inject sample at a voltage between -1 and -5 kV applied
for 10-60s depending on the sample concentration. The cathode
at the capillary inlet was set at -9.5 kV, while the anode at the
capillary outlet was at an electrospray ionization voltage of +2.5
kV, so that the total separation voltage across the capillary was
12kV. Eluents fromCE separations were interfaced toan ion trap
storage reflectron time-of-flight mass spectrometer (CE-IT-reTOF
MS)27-30for mass and sequence analysis of the peptides. This
device is anonscanning instrument that can respond tothe speed
of the CE separations. It is capable of on-line analysis and CE-
MS/MS of each peptide peak using Windows-based software
developed in-house. The CE-MS/MS was performed according
to previous work.23
MALDI-TOF MS. Each purified protein was alsoanalyzed by
MALDI-TOF MS. The sample was spotted to the MALDI plate
followed by 1 µL of matrix layered on top. The spotted plate was
dried in air before MALDI-TOF analysis. The MALDI matrix was
prepared by diluting saturated R-CHCA (Sigma) solution, made
in 50%(v/v) ACN and 1%(v/v) TFA with the same solution at a
1:4ratio(v/v). Standards were prepared as 1mg/mL angiotensin
I, ACTH 1-17, and ACTH 18-39 (Sigma), which are diluted 100
foldwith deionizedwater. Aliquots of13, 21, and25µL weretaken
fromeach ofthedilutedstandardsolutions andmixedwith matrix
toproduce1mL ofsolutionresulting in∼50fmol ofeach standard
in each well spot.
Peptide masses were measured on a Micromass TofSpec2E
with delayed extraction in the reflectron mode using a nitrogen
laser (337 nm). Peptide mass spectra were internally calibrated
resulting inamass accuracy within25ppm. Thecalibratedspectra
were processed using PeptideAuto (Micromass MassLynx ap-
plication) to obtain experimental masses that were submitted to
theSwissProtdatabasefor proteinidentity. Inthedatabasesearch,
a maximum number of two missed cleavages are allowed and
cysteine is unmodified. Homosapiensis specified for the species.
The pH range is not restricted, and the PTMs allowed include
Met-ox, protein N-terminal acetylation, and phosphorylation. The
monoisotopic mass is applied for the MALDI-TOF and ESI-TOF
data, while the average mass is used for CE-TOF MS data.
ESI-TOF Direct Measurement ofDigest. Halfofeach tryptic
digest was dilutedwith water at aratioof1:10. Thedilutedpeptide
mixture was then infused directly into the LCT at a flow rate of
50 µL/min using a syringe pump (Harvard Apparatus, Holliston,
MA, model AH55-2222). The LCT is set at the same condition as
in section NPS-RP-HPLC/ESI-TOF MS above except that the
desolvation temperature is tuned to 200 °C and the desolvation
gas flow is decreased to 150 L/h to optimize the signal for
(26) Bateman, K. P.; White, R. L.; Thibault, P. Rapid Commun. Mass Spectrom.
1997, 11, 307-15.
(27) Li, M. X.; Liu, L.; Wu, J. T.; Lubman, D. M. Anal. Chem. 1997, 69, 2451-6.
(28) Wu, J.; Qian, M. G.; Li, Z.; Liu, L.; Lubman, D. M. Anal. Chem. 1996, 68,
(29) Ding, L.; Kawatoh, E.; Tanaka, K.; Smith, A. J.; Kumashiro, S. Proc. SPIE
1999, 3777, 144-155.
(30) Purves, R. W.; Li, L. J. Am. Soc. Mass Spectrom. 1997, 8, 1085-1093.
Analytical Chemistry, Vol. 75, No. 22, November 15, 2003
RESULTS AND DISCUSSION
2-D Liquid-Phase Separation. CA1d cell lysates were
fractionated using a2-D liquid separationas described inprevious
work.19,20Proteins wereseparatedinthefirst dimensionaccording
toisoelectric point (pI). Each pI fractionwas thenseparatedusing
NPS-RP-HPLC in the second dimension to generate a 2-D mapof
cellular protein expression as shown in Figure 1. The pH range
of the 2-D separation can extend from 3 to 12; however, a limited
number of lanes (pH 4-8) are displayed in this map. The
application of 1.5-µm nonporous particles greatly increases the
separation efficiency.31,32A direct benefit fromthe high efficiency
is that a good LC separation can be achieved without the loss of
resolution.33-36The NPS-RP-HPLC chromatogram of the Rotofor
fraction with a pH value of 6.03 is shown in Figure 2. A steep
gradient was applied at the beginning and at the end of the
was shallow in the middle of the separation to achieve improved
resolution. The result is that over 70 UV absorption peaks each
corresponding to one or more proteins were observed for each
lane within a 30-min separation. Five proteins marked in Figure
1 were selected for further analysis based on obtaining a range
of hydrophobicity and molecular weight. There was ∼200fmol of
each proteinavailablefor analysis. Several oftheselectedproteins
were also known to be implicated in cancer progression.
The main advantage of 2-D liquid separations is that proteins
remain in the liquid phase throughout separation.19-22,37,38The
result is that purified proteins are able to be collected from the
HPLC eluent for further analysis. The proteins are of sufficient
purity so that they can be injected directly into an ESI-TOF MS
for MW analysis. As shown in Figure 3, the molecular weights of
thymosin ?4and HSP60 were obtained from their deconvoluted
ESI-TOF spectrum. Only one peak is present in the deconvoluted
spectrum. In almost all peaks obtained from the HPLC, there is
either only one protein MW observed or one major protein
accompanied by a second protein of lower abundance. All MW
values obtainedareshowninTable1for thesamples under study.
The determination of an exact MW value is essential for determin-
ing the presence of posttranslational modifications. The MW
values listed in Table 1 show that, in some cases, such as lamin
A, the MW value determined is exactly the same as that expected
from the database; consequently, the protein is probably not
modified. In other cases, such as CK18, the protein MW is
different from that expected, indicating the potential presence of
PTMs that need to be determined by other methods. The ability
to obtain an exact MW of a protein embedded in a gel is possible
(31) Hanson, M.; Unger, K. K.; Mant, C. T.; Hodges, R. S. Trends Anal. Chem.
1996, 15, 102-110.
(32) Banks, J. F.; Gulcicek, E. E. Anal. Chem. 1997, 69, 3973-3978.
(33) Barder, T. J.; Wohlman, P. J.; Thrall, C.; DuBois, P. D. LC-GC, 1997, 69,
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C. G. J. Chromatogr., B 2002, 782 (1-2), 111-125.
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Figure 2. HPLC chromatogram (solid line) for the Rotofor fraction,
pH 6.03. The dotted line represents the profile of the pH gradient,
where the % B value is the percentage of acetonitrile in the mobile
phase during elution. In both mobile phases A and B, 0.1% TFA was
added. The separation is monitored at 214 nm.
Figure 1. IEF-NPS RP HPLC 2-D map of Ca1DCL1 cell lysate.
Analytical Chemistry, Vol. 75, No. 22, November 15, 2003
using a number of methods described in the literature8,9,39but
requires substantial sample preparation and a significant amount
of the sample may be lost during recovery. Further, the proteins
destaining, andpurificationas inthecaseof2-D gels. Thedigested
proteins are also of sufficient purity so that they can be directly
analyzed with minimal sample preparation by either CE-TOF MS
or MALDI-TOF MS for sequence analysis or identification of
PTMs. It should be emphasized that the proteins being analyzed
in this work are samples that have been extracted from whole-
cell lysates as opposed to purified proteins obtained from a
Capillary Electrophoresis TOF Mass Spectrometry. One
of the key advantages of this method is that the high resolution
and speed of CE-TOF MS24-26,40-45can be used for analysis of
the protein digests of the proteins obtained from the 2-D liquid
separations. This can be performed with minimal sample prepara-
tion. After tryptic digestion of the proteins purified by 2-D liquid-
phase separation, the digest solution is completely dried down
and then reconstituted with 1 µL of water. The separation
efficiency of CE even for large proteins is shown in the elution
profile of the HSP60 tryptic digest shown in Figure 4. This
separation is performed in less than 10 min with only 3 fmol of
sample injected into the capillary. The peaks are reasonably well
separated. The mass spectra shown in the insets (Figure 4)
suggest that only limited coelution occurs. Although very little
sample preparation has been performed prior to CE-TOF MS
analysis, the efficiency of the CE separation and the quality of
ESI-MS spectra are not affected significantly even for these
proteins isolated out of genuine cellular environments. Thus, the
(39) Cohen, S. L.; Chait, B. T. Anal. Chem. 1997, 247, 257-267.
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C.; Lee, H. G. Anal. Chem. 1997, 69 (16), 3205-3211.
(43) Fang, L.; Zhang, R.; Willams, E. R.; Zare, R. N. Anal. Chem. 1994, 66, 3696-
(44) Hsieh, F.; Baronas, E.; Muir, C.; Martin, S. A. RapidCommun. MassSpectrom.
1999, 13 (1), 67-72.
(45) Cao, P.; Moini, M. J. Am. Soc. Mass Spectrom. 1998, 9 (10), 1081-1088.
Figure 3. Deconvoluted ESI-TOF spectra of (A) thymosin-?4and
(B) HSP60. The intact molecular weight of the proteins are obtained.
The peak at 28 983 in (B) is a harmonic peak generated during
deconvolution; it is half of the molecular weight of 57 966.
T able 1. Comparisons between T heoretic al and
Experimental Molec ular Weights
theor protein name
60-kDa heat shock proteina
thymosin ?4, N-terminal
aThis protein is the protein part of unprocessed precursor (mito-
chondrial 60-kDa heat shock protein). The molecular weight of the
truncated form, in which the first 26aminoacids are missed, is 57 963.
bThe molecular weight includes N-terminal acetylation of thymosin
Figure 4. CE-MS elution profile of tryptic digest of heat shock
protein with inset mass spectra of (a) ALMLQGVDLLADAVAVTMGPK
(38-58) at m/z 705.67, (b) LNER (390-393) at m/z 266.29, and (c)
KDR (418-420) at m/z 209.77.
Analytical Chemistry, Vol. 75, No. 22, November 15, 2003