Comparative Identification of Prostanoid Inducible Proteins by LC-ESI-MS/MS and MALDI-TOF Mass Spectrometry

Comparative Identification of Prostanoid InducibleProteins by LC-ESI-MS/MS and MALDI-TOF Mass

Spectrometry

Maria D. Person, Herng-Hsiang Lo, Kelly M. Towndrow,† Zhe Jia,Terrence J. Monks, and Serrine S. Lau*

Center for Molecular and Cellular Toxicology, Division of Pharmacology and Toxicology,College of Pharmacy, The University of Texas at Austin, Austin, Texas 78712

Received June 21, 2002

Protein identification by MS is well-established. Mixtures of proteins from cell extracts areseparated by either one- or two-dimensional gel electrophoresis, and specific bands or spotsare subjected to in-gel digestion and subsequent analysis by MS. The two most common typesof ionization used in MS are electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI). When ESI is used, the sample is typically analyzed by inline HPLC-ESI-MS/MS with fragmentation of individual digest peptides, followed by database comparisonbetween theoretical and experimental fragmentation patterns. MALDI-MS analysis is basedon peptide mass mapping, with mass measurements of the digest peptides searched against adatabase of theoretical digests. We give here the results of a comparison between ESI-ion trapand MALDI-TOF (time-of-flight) analysis of 11-deoxy,16,16-dimethyl prostaglandin E2 (DDM-PGE2) inducible proteins. Individual peptides identified by the two techniques differed, ingeneral, but the resulting protein identification was the same. Slightly higher coverage of eachprotein was obtained by MALDI-TOF, but the MS/MS data were more definitive by requiringfewer peptides to assign a positive identification. Both methods effectively identified twoproteins in the same gel band. The samples here are derived from a renal epithelial cell line(LLC-PK1) established from the New Hampshire minipig, a species poorly represented in thecurrent database, and strategies and limitations for analyzing such species are discussed.

Introduction

Changes in protein expression and/or function havelong been appreciated in environmental chemical-inducedtoxicity and cancer. For many years, however, profilingprotein targets of toxic chemicals has been a nearlyimpossible task. Recent developments in MS ionizationmethods and instrumentation now make possible therapid, high throughput analysis and identification ofproteins. MS has become the method of choice forsequencing peptides and proteins in samples with limitedamounts of protein available. These analytical capabili-ties have driven the growth of proteomics, the study ofthe protein complement of the genome. The explosion inproteomics research has exposed an increasing numberof biological and medical researchers to the technique ofprotein identification by MS (1-6). Differential proteinexpression between wild-type and transgenic animals,and proteins differentially expressed as a consequenceof toxicant exposure, can be visualized by one- or two-dimensional gel electrophoresis of the appropriate samples.Similarly, antibody staining of gels can reveal specificproteins modified by toxicants. To identify a protein inthe gel, N-terminal Edman sequencing or mass spectro-metric techniques are used. Increased instrumentalsensitivity and the development of suitable preparativetechniques for use with mass spectrometers have in-

creased the popularity of this approach. Currently, themost widely utilized protocol for proteins separated bySDS-PAGE uses the protease trypsin to digest theproteins in the gel, and then, the digested peptides aresubsequently extracted and analyzed by MS. The proteinsare identified by either peptide mass mapping or peptidesequencing followed by a database search (Figure 1).

Peptide mass mapping utilizes a list of tryptic peptidemasses derived from a spectrum to query a database oftheoretical digests and identifies the unknown proteinfrom the best match (7-9). This technique is usedpredominately with MALDI-TOF1 mass spectrometers.

* To whom correspondence should be addressed. Tel: 512-471-5190.Fax: 512-471-5002. E-mail: [email protected].

† Present address: Department of Investigative Toxicology, LillyResearch Laboratories, Eli Lilly and Company, Greenfield, IN 46140.

Figure 1. Alternative strategies for protein identification usingMS.

757Chem. Res. Toxicol. 2003, 16, 757-767

10.1021/tx020049d CCC: $25.00 © 2003 American Chemical SocietyPublished on Web 05/09/2003

The main advantage is high throughput, as spectralacquisition times are only seconds per sample. Currentinstrumentation, employing delayed extraction and re-flectron detectors, has the mass accuracy necessary fordefinitive protein identification with ever-increasingdatabase size (10). An alternative approach is that of MS/MS fragmentation, or peptide sequencing, which isolatesand fragments individual tryptic peptides and thenqueries a database of expected fragmentation ions oftryptic peptides (11, 12). When ESI is used for ionization,fragmentation is accomplished by collision-induced dis-sociation (CID), where collisions with an inert gas, suchas helium, cause the peptides to fragment into charac-teristic product ions. Alternatively, PSD fragments aredetected when MALDI-TOF is used. For separation of thepeptides, reversed phase HPLC can be coupled in-line tothe ESI mass spectrometer. The analysis requires in-creased time for the HPLC run, and the MS/MS databasesearch requires more computer power to search the manyspectra generated during the run and to correlate theresults. The results of an MS/MS-based search are morelikely to identify a component present at relatively lowlevels, or with few tryptic peptides, when a mixture ofproteins is present. Additionally, because sequence in-formation is generated from MS/MS spectra, similarity-based searches can identify proteins not present in thedatabase for the species under study.

A number of database search engines are publiclyavailable on the web, or for license, such as Pro-tein Prospector (prospector.ucsf.edu), Mascot (www.matrixscience.com), and Prowl (prowl.rockefeller.edu) aswell as proprietary software from mass spectrometermanufacturers. The search engines can be used to querydifferent protein and translated DNA databases, whichare continually updated. The NCBI and Swiss-Protdatabases are most commonly used, with Swiss-Protproviding extensive annotation and NCBI containingmore sequence variants. The database sequences gener-ally represent precursor proteins and do not includeposttranslational modifications or processing. Some pro-teins in the database are present only as sequencefragments, as the full sequence has yet to be determined.

While both MALDI-TOF peptide mapping and HPLC-ESI-MS/MS techniques have been used successfully toidentify proteins, there has been little direct comparisonof the relative effectiveness of the two techniques. Fem-tomolar sensitivity is possible with either techniquedepending on instrument choice. In a number of studies,MALDI-MS has been used for initial analysis, and ESI-MS/MS has been used on samples that are not identifiedin the initial MALDI screen (13, 14). This strategy takesadvantage of the high throughput of MALDI and themore definitive results of ESI-MS/MS analysis. When theresults of an analysis of a single HPLC fraction contain-ing tryptic digest peptides from an in-gel digest werecompared by nanospray ESI and MALDI, the resultswere complementary, with two proteins identified by bothmethods (15). However, the superior sequencing ability

of the nanospray ESI-QTOF instrument allowed for theidentification of two additional protein components.When a set of yeast proteins separated by two-dimen-sional gel electrophoresis was subjected to in-gel digestand analysis by MALDI-TOF and LC-ESI-MS/MS (16),90% of the proteins were successfully identified byMALDI and 100% by ESI. A study of yeast membraneproteins separated by one-dimensional gel electrophoresisidentified more proteins by nano-LC-ESI-MS and -MS/MS than by MALDI-MS and -PSD, using slightly differ-ent sample preparation methods for the different digests(17). Because the yeast genome sequence is complete, thedatabase search should be able to identify every proteinin the sample. This is not the case for most other species,where the genome sequence is incomplete. In the case ofmammals, only the rat, mouse, and human are well-represented, and the genome and protein product varia-tions are considerably more complex.

We here provide an example of the applicability ofcombined MALDI-TOF-MS and HPLC-ESI-ion trap-MS/MS analysis for solving specific questions that addressthe mechanism of chemical or toxicant action. We makea broad comparison of the results of protein identificationof in-gel digest samples using protocols and instrumentsin widespread usage, MALDI-TOF-MS, and HPLC-ESI-ion trap-MS/MS. While not representing the most so-phisticated instruments available, these systems arecurrently available with extensive automation, withspectral acquisition, and with processing methods ableto generate protein identification lists with little userintervention required after sample loading. The proteinsanalyzed were derived from DDM-PGE2-treated LLC-PK1

cells (New Hampshire minipig renal proximal tubular cellline). DDM-PGE2 is a synthetic analogue of prostaglandinE2 and provides protection to LLC-PK1 cells against thereactive oxygen species generating toxicant 2,3,5-tris-(glutathion-S-yl)hydroquinone (18). Differential proteinexpression was utilized to identify proteins induced byDDM-PGE2 and thus elucidate the potential mechanismby which it provides cytoprotection (19). DDM-PGE2

selectively stimulated the synthesis of several proteinsin LLC-PK1 cells, as determined by [35S]methioninelabeling. These proteins, which likely contribute to thecytoprotective effects of DDM-PGE2, were identified byMS and confirmed by western blot analysis (19). Thecomparative mass spectral approaches described hereinshould have applicability to unraveling mechanisms ofchemical-induced toxicities when those toxicities areassociated with functional changes in the proteome. Suchchanges in protein expression profiles may result from(i) chemical exposure, (ii) comparisons between trans-genic/knockout and wild-type littermates, (iii) effects ofusing dominant negative and other transfection technolo-gies, and (iv) comparisons between normal and neoplasticor diseased tissue.

Experimental Procedures

Chemicals. HPLC grade solvents were purchased from EMScience (Cincinnati, OH), acetic acid was purchased fromAldrich (Milwaukee, WI), and other reagents were purchasedfrom Sigma (St. Louis, MO).

Protein Extraction from Cells. Details of the samplepreparation are given in Towndrow et al. (19). Post confluentLLC-PK1 cells were exposed to 1 µM DDM-PGE2 or 0.04% (v:v)ethanol (vehicle control) in 35S-methionine-containing mediumfor 24 h. At the end of the experiment, cells were pelleted and

1 Abbreviations: ACN, acetonitrile; DDM-PGE2, 11-deoxy,16,16-dimethyl prostaglandin E2; EF, elongation factor; ESI, electrosprayionization; GRP, glucose-regulated protein; HSP, heat shock protein;MALDI, matrix-assisted laser desorption/ionization; MS/MS, tandemmass spectrometry; NCBI, National Center for Biotechnology Informa-tion; PSD, post-source decay; QTOF, quadrupole time-of-flight; TER,transitional endoplasmic reticulum; moesin, membrane-organizingextension spike protein; TOF, time-of-flight; Xcorr, raw cross-correla-tion score.

758 Chem. Res. Toxicol., Vol. 16, No. 6, 2003 Person et al.

lysed, and the supernatant was collected. 35S-Methionine-labeledproteins were separated by SDS-PAGE, stained with Coo-massie Blue R, dried, and analyzed by autoradiography. Eightbands of interest were identified by increased radioactivity ascompared to the control. Separate gels were prepared foranalysis by MS.

In-Gel Protein Tryptic Digestion. About 600 µg of proteinlysate from DDM-PGE2-treated cells was loaded onto four lanesof a single gel. Proteins of interest were selected, and theidentical bands were cut from all four lanes. Half of the samplewas digested and subjected to LC-MS/MS analysis. The remain-ing half was stored at -80 °C in a 5% acetic acid solution andlater subjected to in-gel digestion followed by MALDI-MSanalysis. In-gel tryptic digestion of selected bands was basedon a modification of standard protocols (20, 21). Prior to in-geldigest, individual bands were cut into 1 mm pieces anddestained in 5% acetic acid, 50% methanol to remove theCoomassie Blue. Gel pieces were dehydrated with ACN, andresidual ACN was evaporated in a SpeedVac (Therma Savant,Hollowbrook, NY). Proteins were then reduced with 10 mM DTTin 100 mM NH4HCO3 at room temperature for 1 h. ResidualDTT was removed, and cysteines were alkylated with 50 mMiodoacetamide (in 100 mM NH4HCO3) for 1 h. After the residualiodoacetamide was removed, gel pieces were subjected twice towashing (100 mM NH4HCO3 for 10 min) and dehydration (5min in ACN). Gels were dried for 2-3 min in a SpeedVac andrehydrated on ice with 20 ng/µL Sequencing Grade ModifiedTrypsin (Promega, Madison, WI; in 50 mM NH4HCO3) for 10-15 min. Excess trypsin was removed, 20 µL of 50 mM NH4HCO3

was added, and gel pieces were digested overnight at 37 °C.After they were digested, peptides were extracted twice in 75µL of 5% formic acid/50% ACN. The samples were reduced involume to 10-20 µL with a SpeedVac.

MALDI-TOF Analysis. Prior to MALDI-MS analysis, thein-gel digests from bands 1 and 2 (Figure 2) were desalted usinga C18 0.6 µL ZipTip (Millipore, Bedford, MA), with 0.1% formicacid in water as the equilibration buffer and 0.1% formic acidin 50% ACN as the elution buffer, following the manufacturer’sprotocol. MALDI-TOF spectra were taken on the delayedextraction Voyager De-Str (Applied Biosystems, Framingham,

MA) instrument equipped with a 2 m linear flight path usingthe reflector detector in the positive ion mode. The instrumentwas equipped with a nitrogen laser operating at 337 nm.Standard method parameters were used, with a 200 ns delaytime. The low mass gate was set at 600, and spectra wereacquired over the mass range of 700-3600 Da. The matrix usedwas R-cyano-4-hydroxycinnamic acid (Agilent, Palo Alto, CA),mixed 1:1 with the sample and drop dried on a gold target in atotal volume of 1 µL. A mixture of Bio-Rad (Hercules, CA) czestandards at 1 ng/µL and ACTH (Sigma) at 2.5 ng/µL was usedfor external calibration of the MALDI spectra, and 64 spectrawere averaged per sample. The peptide mass list was derivedfrom the monoisotopic peaks with a signal-to-noise ratio of atleast two in the range of 790-3600 Da.

Peptide mass lists were filtered for removal of trypsinautolysis peaks, low mass matrix-related ions, and peptideadducts of sodium (+22) and potassium (+38). The mass listfor each sample was entered in the search program, MS-Fit, inthe Protein Prospector suite (prospector.ucsf.edu). The Swiss-Prot and Owl databases were searched using a 75 ppm peptidemass tolerance based on external calibration of a nearby spot,a maximum of two missed cleavages, and carbamidomethylationof the cysteines. For the database search, the protein molecularmass range was restricted to 1000-100 000 Da and the searchwas performed both for all species and for all mammals. Forthe two high molecular mass bands, a mass tolerance of 50 ppmwas used with external calibration at a position adjacent to thesample spot on the MALDI target and the protein molecularmass range was not restricted. The protein represented by thehighest scoring match was reported, and peptide sequences wereassigned according to the best match. For samples containingtwo proteins, the second protein was not the highest in scorebut provided a minimum of three matching peptides and similarprotein molecular mass.

LC-ESI-MS/MS Analysis. A microbore HPLC system (Magic2002, Michrom BioResources, Auburn, CA) coupled in-line withan electrospray ion trap mass spectrometer (LCQ, ThermoFinni-gan, San Jose, CA) was used for the LC-ESI-MS/MS analyses.Peptides were separated with a 0.5 mm × 50 mm MAGIC MSC18 column (5 µm particle diameter, 200 Å pore size) usingmobile phases A (ACN:water:acetic acid:trifluoroacetic acid,2:98:0.1:0.02) and B (ACN:water:acetic acid:trifluoroacetic acid,90:10:009:0.02). A linear elution gradient of 5-65% B over 30min was followed by 95% B for 5 min at a flow rate of 20 µL/min. Mass spectra were accumulated over a 45 min run timetriggered by the start of the HPLC run. Automated acquisitionof MS and MS/MS spectra was executed by data-dependentscanning using ThermoFinnigan Excalibur software. The set-tings for the LCQ were as follows: spray voltage, 3.5 kV;capillary temperature, 200 °C; capillary voltage, 46 V; tube lensoffset, 55 V. The scan time settings used were 200 ms maximuminjection time for all scans, with averaging of three microscansfor full scan and five microscans for Zoom and MS/MS scans.The target number of ions was 1e8 for full scan, 1e7 for Zoomscan, and 2e7 for MS/MS. The full scan range for MS was 200-2000 Da. Data-dependent MS/MS acquisition was performedwith a default charge state of 2, isolation width of 2 amu,normalized collision energy level at 35%, and required a minimalsignal of 30 000 counts. The scan event sequence included onefull scan followed by a dependent Zoom scan and MS/MS scanof the most intense ion seen in the full scan.

Sequences of individual peptides were identified using theSEQUEST algorithm, incorporated into the ThermoFinniganBIOWORKS software, to correlate the MS/MS spectra withamino acid sequences in either the NCBI nonredundant FASTAor OWL protein database. The search was conducted for humanor all species, using trypsin digest and one missed cleavagemaximum. MS/MS data were searched for charge states +1, +2,and +3. The protein identified as the best hit and having themost peptide matches was reported, along with secondarymatches. Peptides reported were those ranked first or secondhaving greater than 50% of the expected fragment ions, gener-

Figure 2. Effect of DDM-PGE2 on the induction of specificproteins in LLC-PK1 cells. LLC-PK1 cells were exposed to 1 µMDDM-PGE2 (D) or to ethanol vehicle control (E) for 24 h in thepresence of 35S-methionine. Protein lysates (1 × 106 CPM/lane)from 35S-labeled cells were collected and then separated viaSDS-PAGE and detected by autoradiography. Eight bands wereselected for protein identification by MS.

Protein Identification by LC-ESI-MS/MS and MALDI-TOF Chem. Res. Toxicol., Vol. 16, No. 6, 2003 759

ally with preliminary Sp scores greater than 200 and Xcorrvalues greater than 1.

Results and DiscussionMALDI and ESI Give Identical Protein Identifi-

cation Results. Samples from a single gel were analyzedindependently via MALDI-MS or HPLC-ESI-MS/MS. Theeight bands were selected for in-gel digestion and iden-tification based upon differential protein induction be-tween DDM-PGE2-treated and untreated LLC-PK1 cells(Figure 2). Protein identification experiments were

repeated several times by LC-MS/MS as described inTowndrow et al. (19). MALDI-TOF was used for peptidemass mapping, with a typical spectra shown in Figure 3where GRP 78 was identified from 13 tryptic peptidemasses. The mass lists derived from the MALDI-TOFspectra for each sample are shown in Table 1 with theidentified masses shown in bold or italics. The samesamples were also analyzed by HPLC-ESI-MS/MS. Fig-ure 4 illustrates a typical MS/MS spectrum showing thefragmentation of a single tryptic peptide from the proteinfilamin 1. The daughter ions most commonly seen are

Figure 3. MALDI-MS identification of GRP 78. The MALDI-MS spectrum of the in-gel digest of gel band 5 is shown, with the GRP78 tryptic peptides labeled. GRP 78 peptide fragment numbering is based on tryptic cleavage of the processed human GRP 78 sequence.

Table 1. MALDI-MS Mass Lists for Each In-Gel Digest Banda

gel band band 1 band 2 band 3 band 4 band 5 band 6 band 7 band 8

mass MH+ 1007.52 1449.79 1172.69 1194.64 986.51 1045.54 1025.58 795.481045.56 1571.86 1274.69 1236.63 1191.64 1104.59 1286.81 945.561180.57 1722.80 1329.71 1264.65 1435.74 1182.59 1306.60 976.451225.59 1773.78 1361.69 1348.67 1460.75 1233.59 1314.70 1132.511285.73 1777.81 1402.79 1513.77 1512.69 1310.68 1341.68 1171.531298.72 1950.00 1554.74 1589.87 1528.77 1578.91 1352.66 1198.711358.64 2045.04 1599.82 1778.93 1566.77 1928.90 1380.67 1499.681400.66 2166.10 1742.84 1782.96 1642.81 2081.96 1404.70 1515.721413.67 1770.86 1786.96 1677.81 1588.84 1673.761415.69 1799.89 1808.96 1815.94 1779.89 1790.871429.70 1852.93 1911.04 1887.92 1996.91 1954.011489.80 1887.97 2015.01 1933.96 2432.07 1960.901505.76 1951.91 2255.97 2015.97 2531.27 2214.941518.80 2090.12 2018.94 2686.99 2230.921529.83 2143.02 2083.00 3147.12 3183.441533.80 2220.11 2164.90 3199.531561.81 2256.051595.83 2353.171616.77 2498.131923.95 2639.152343.06 2817.262415.10 3117.432749.372893.51

% matchedb 50 38 68 100 81 75 40 88primary ID filamin 1 myosin EF-2 HSP 90 â GRP 78 moesin EF-1-R â,γ-actinsecondary ID TER-ATPase HSP 90 R R-actina Those matched to primary proteins are shown in bold, and those matched to secondary proteins are shown in italics. b Percentage of

masses identified out of those submitted.


those produced by cleavage of the peptide amide bond,with charge retention at the N-terminal designated b ionsand charge retention at the C-terminal designated y ions(22). The mass difference between two ions in a givenseries is the amino acid residue mass. The set of all MS/MS spectra collected over one HPLC run was submittedto the SEQUEST database search program, and theresults from the best match are shown in Table 2. Asummary of peptides identified for each protein is shownin Table 2. The ESI assignment is based on MS/MSfragmentation spectra whereas the MALDI assignmentsare based on peptide mass. In all cases where a matchwas made, the MALDI and ESI methods identified thesame proteins, even when two proteins were present inthe sample.

MALDI and ESI Give Different Amino Acid Cov-erage. Proteins were identified from a minimum of threepeptides for each gel band. The results were confirmedby multiple LC-ESI-MS/MS runs (19). The averagenumber of peptides identified from their MS/MS frag-mentation pattern was 7.5 per protein, giving an averageamino acid coverage of 12%. A second set of samples wassubjected to in-gel digest and independently analyzed byMALDI-MS peptide mapping. Here, the average numberof peptides per protein is 8.7, with 17% amino acidcoverage (not including gel band 7 for which there is nocorresponding ESI data). There were 11 proteins identi-fied in the eight bands. The average number of identicalpeptides seen in both the MALDI and the ESI analysesis 2.7. While the samples came from the same gel, thein-gel digests were performed separately, accounting forsome differences in peptides produced. However, becauseeach method gave amino acid coverage of <20% onaverage, it is not surprising that the overlap is minimal.Peptide coverage is lower in this particular case becausethe species studied, minipig, is poorly represented in thedatabase, and most of the proteins were identified fromthe sequence of another mammalian species.

The two analytical methods have differing detectionefficiencies for the peptides produced by the tryptic

digest. In a MALDI spectrum, the low mass regionusually is dominated by matrix peaks and thus is notused in the database search. This eliminates peptidesshorter than seven or eight amino acids. In contrast, theESI ion trap is optimized for peptides between m/z valuesof 300 and 2000 Da and can detect shorter peptides.Because the longer peptides usually involve doubly ortriply charged species and the charge state of the frag-ment ions is not resolved, the SEQUEST search algo-rithm has more difficulty making the correct assignmentfor peptides greater than 20 amino acids in length. Them/z limit of 2000 precludes detection of high mass/chargefragment ions, and thus, it is more difficult to accuratelymatch the data to a theoretical spectrum. The columnand gradient conditions for the HPLC separation alsoprovide better separation and thus detection for theintermediate length peptides. The MALDI peptides arelonger on average than those that are detected by ESI;thus, the amino acid coverage is slightly greater forMALDI. However, the ESI analysis includes only thosepeptides definitively identified via MS/MS fragmentation,whereas the MALDI identification is by mass alone. TheMS/MS data contain sequence information; thus, fewerpeptides are needed for conclusive identification. Theanalysis highlights the complementary nature of the twotechniques that can prove useful when trying to maxi-mize coverage. However, it is also clear that even usingboth of these conventional methods gives much less than100% coverage.

In either case, the number of peptides did not correlatewith protein size. While large proteins have more trypticpeptides than small proteins, the ability to detect thepeptides is more a function of protein amount and thequality of the sample preparation. While staining of thegels shows the amount of protein present by mass, themolar amount is more important for detection of peptides.Larger proteins thus require more sample to achieve thesame level of peptide concentration. Sample cleanupusing ZipTip also significantly enhanced the detection ofpeptides obtained from in-gel digest by MALDI. Reducing

Figure 4. MS/MS identification of filamin 1. An MS/MS spectrum identified as the peptide VTVLFAGQHIAK in filamin 1 from gelband 1. The doubly charged parent ion has m/z of 646.6, and the singly charged b and y fragment ions are labeled. From the differencein mass for adjacent members of the y ion series y4 to y10, the amino acid sequence can be established.


Table 2. Summary of Results of ESI and MALDI Analyses of In-Gel Digest Bandsa

bandmol mass

(kDa)peptide sequence from

ESI (Xcorr)AA (%)b

overlapcpeptide sequence from

MALDIAA (%)b

overlapc protein

1 281 LVSIDSK (1.2)AIVDGNLK (1.1)AGVAPLQVK (1.0)YTILIK (1.2)GAGTGGLGLAVEGPSEAK (3.1)VTVLFAGQHIAK (3.7)LLGWIQNK (2.1)DAGEGLLAVQITDPEGKPK (3.3)LIALLEVLSQK (1.7)

3.70/9

IQQNTFTREATTEFSVDARLPQLPITNFSRYGGDEIPFSPYRWGDEHIPGSPYRAFGPGLQGGSAGSPARFVPAEMGTHTVSVKd

SPFSVAVSPSLDLSKe

AEAGVPAEFSIWTRe

AYGPGIEPTGNMVKKWCNEHLKCVSKRAGQSAAGAAPGGGVDTRDAEMPATEKe

VANPSGNLTETYVQDRGDGMYKe

VGSAADIPINISETDLSLLTATVVPPSGR

6.70/14

filamin 1 orendothelialactin-bindingprotein(ABP-280)f

2 227 VIQYLAHVASSHK (3.6)VSHLLGINVTDFTR (2.8)LQQELDDLLVDLDHQR (4.8)VISGVLQLGNIVFKK (2.2)QLLQANPILEAFGNAK (3.7)VISGVLQLGNIVFK (3.2)

3.82/6

VSHLLGINVTDFTRQEEEMMAKEEELVKLQQELDDLLVDLDHQR

2.22/3

myosin,heavy chain,nonmuscletype Af

3 95 NPADLPK (1.5)LDSEDKDKEGKPLLK (2.5)VFSGLVSTGLK (1.8)AYLPVNESFGFTADLR (4.0)

5.71/4

EDLYLKPIQRKEDLYLKPIQRYLAEKYEWDVAEARAYLPVNESFGFTADLRFYAFGRVFSGVVSTGLKVRARPFPDGLAEDIDKGEVSARALLELQLEPEELYQTFQRGVQYLNEIKDSVVAGFQWATKSDPVVSYRETVSEESNVLCLSKDGSGFLINLIDSPGHVDFSSEVTAALRLMEPIYLVEIQCPEQVVGGIYGVLNRK

231/11

EF-2f

3 89 VALLK (1.3)HPALFK (1.3)ELQELVQYPVEHPDK (3.1)NAPAIIFIDELDAIAPK (5.0)

5.30/4

GILLYGPPGTGKWALSQSNPSALRGPELLTMWFGESEANVRd

AVANETGAFFFLINGPEIMSK

7.70/4

TER-ATPaseg

4 83 LGIHEDSTNR (2.8)VVVITK (1.9)YESLTDPSK (1.5)SLVSVTK (0.97)SIYYITGESK (1.7)SLTNDWEDHLAVK (4.6)GVVDSEDLPLNISR (3.7)ALLFIPR (1.7)HSQFIGYPITLYLEK (5.9)

132/9

IDIIPNPQERRAPFDLFENKHFSVEGQLEFRGVVDSEDLPLNISRHLEINPDHPIVETLRHSQFIGYPITLYLEKKHLEINPDHPIVETLRVILHLKEDQTEYLEERHNDDEQYAWESSAGGSFTVR

152/9

HSP 90 â;HSP 84f

4 85 VTVITK (1.2)LGIHEDSQNR (1.3)TLVSVTK (1.6)SLTNDWEDHLAVK (4.6)GVVDSEDLPLNISR (3.7)HLEINPDHSIIETLR (4.1)HSQFIGYPITLFVEK (5.1)

113/7

APFDLFENRKRAPFDLFENRHFSVEGQLEFRGVVDSEDLPLNISRELHINLIPNKQDRHSQFIGYPITLFVEKHLEINPDHSIIETLRVILHLKEDQTEYLEERHNDDEQYAWESSAGGSFTVR

163/9

HSP 90 R;HSP 86g

5 72 LIGDAAK (1.6)LIPR (1.3)IQQLVK (1.7)VLEDSDLK (1.9)LTPEEIER (1.0)TKPYIQVDIGGGQTK (3.7)NQLTSNPENTVFDAK (4.1)FLPFK (1.3)SQIFSTASDNQPTVTIK (5.1)ITPSYVAFTPEGER (3.1)VTHAVVTVPAYFNDAQR (4.1)IINEPTAAAIAYGLDKR (5.3)IEWLESHQDADIEDFK (1.5)DNHLLGTFDLTGIPPAPR (5.6)

266/14

LTPEEIERVYEGERPLTKVMEHFIKLYKKSDIDEIVLVGGSTRAKFEELNMDLFRAKFEELNMDLFRd

ITPSYVAFTPEGERNQLTSNPENTVFDAKIINEPTAAAIAYGLDKRVTHAVVTVPAYFNDAQRDNHLLGTFDLTGIPPAPRKVTHAVVTVPAYFNDAQRIEIESFYEGEDFSETLTR

246/13

78 kDa GRP78; BiPh

6 68 ESPLLFK (1.1)LFFLQVK (2.0)EVWFFGLQYQDTK (3.1)

4.70/3

ALTSELANARIGFPWSEIRAPDFVFYAPRIQVWHEEHRKAPDFVFYAPRFYPEDVSEELIQDITQR

9.70/6

membrane-organizingextensionspike protein(moesin)g


the amount of salt and detergent aids in crystallizationof the MALDI samples and prevents salt adduct ions.

Both Techniques Identify Two Proteins in aSingle Band. Three of the eight gel bands contained twodetectable proteins. MALDI and ESI were both able toidentify the two components and to distinguish between

proteins with similar sequences, such as HSP 90 R andâ in band 4, as well as identify the unrelated proteinsEF-2 and TER-ATPase in band 3. Figure 5 shows theMALDI-MS spectrum of sample 4, containing HSP 90 Rand â. While many peptides are present in both isoforms,there are several peptides identified that belong to a

Table 2 (Continued)

band

molmass(kDa)

peptide sequence fromESI (Xcorr)

AA (%)b

overlapcpeptide sequence from

MALDIAA (%)b

overlapc protein

7 50 0 IGGIGTVPVGREHALLAYTLGVKYYVTIIDAPGHRTHINIVVIGHVDSGKFETSKYYVTIIDAPGHRVETGVLKPGMVVTFAPVNVTTEVKd

170/6

EF-1R-1f

8 42 AGFAGDDAPR (1.6)LDLAGR (1.5)IIAPPER (1.7)QEYDESGPSIVHR (3.2)RGILTLK (1.2)GILTLK (1.1)IWHHTFYNELR (3.6)AVFPSIVGRPR (2.9)VAPEEHPVLLTEAPLNPK (4.3)SYELPDGQVITIGNER (4.8)

267/10

IIAPPERAGFAGDDAPRGYSFTTTAERHQGVMVGMGQKAVFPSIVGRPRQEYDESGPSIVHRi

IWHHTFYNELRSYELPDGQVITIGNERVAPEEHPVLLTEAPLNPKDLYANTVLSGGTTMYPGIADRDLYANTVLSGGTTMYPGIADRd

TTGIVMDSGDGVTHTVPIYEGYALPHAILRTTGIVMDSGDGVTHTVPIYEGYALPHAILRd

427/13

actin,cytoplasmic 1(â-actin)h

actin,cytoplasmic 2(γ-actin)f

8 42 AGFAGDDAPR (1.6)LDLAGR (1.5)IIAPPER (1.7)RGILTLK (1.2)GILTLK (1.1)IWHHTFYNELR (3.6)AVFPSIVGRPR (2.9)YPIEHGIITNWDDMEK (4.4)SYELPDGQVITIGNER (4.8)

226/9

IIAPPERAGFAGDDAPRHQGVMVGMGQKAVFPSIVGRPRIWHHTFYNELRSYELPDGQVITIGNERYPIEHGIITNWDDMEK

226/7

actin,R-skeletalmuscle(R-actin 1)g

a Peptides derived from ESI are sequenced via MS/MS fragmentation. MALDI peptides are matched by peptide mass only. Peptidesunderlined are those seen in both analyses. b Amino acid coverage (%). c Number of identical peptides seen in both ESI and MALDI/totalnumber of peptides seen by either ESI or MALDI. d Peptide with oxidized methionine species. e Peptide pairs with identical mass cannotbe distinguished in MALDI. f Pig sequence is not present in the database. g Complete pig sequence in the database. h Pig sequencefragment(s) in database. i Peptide has pyroglutamate modification.

Figure 5. MALDI-MS identification of HSP 90 R and HSP 90 â in band 4. The HSP 90 peptide peaks are labeled according to theisoform(s) that contain each peptide, and trypsin autolysis peaks are labeled. Other prominent peaks are salt adducts of peptides.


single isoform. Data from the LC-ESI-MS/MS on thecorresponding sample are shown in Figure 6. The basepeak chromatogram from the entire 45 min run is shown,where peptides eluted from 7 to 34 min. The MS/MSspectra show two peptides from the â and R isoforms ofHSP 90, respectively, and the difference of two aminoacids is easily identifiable from the fragmentation b andy ion series. In this case, the proteins in the mixture areequally abundant and are thus readily identified. Asmore peptides are required for MALDI identification, itshould be easier to identify multiple proteins via ESI-MS/MS. The superior MS/MS data quality of nanosprayMS/MS on a QTOF can identify protein components inlower abundance than can be identified by MALDI-MS(15). The additional step of HPLC separation of peptidesalso aids in the identification of multiple proteins. Ineither case, secondary proteins may be easily overlookedwhen the dominant source of signal is identified, as theymay appear much lower in the list of protein hits. Searchengines that allow the remaining unmatched peptidemasses to be resubmitted for a search after the dominantprotein is selected can help to identify secondary proteinsin MALDI-MS (MS-FIT for example).

The ability to look at all of the peptides simultaneouslyin a MALDI-MS spectrum allows a qualitative pictureof the completeness of the identification. As Figure 3shows, when most of the masses can be matched, thereis clearly a dominant protein in the band. In Figure 5,there are two proteins of equal abundance, HSP 90 R andâ, which give similar ion intensity in the spectra.

Proteins Can be Identified When a Species’ Pro-tein Sequence Is Not in the Database. The samplesinvestigated were derived from the New Hampshireminipig, a species nonexistent in the database. Thenearest relative, Sus scrofa, is also poorly represented.Only 768 of the 100 225 protein sequences in the Swiss-Prot database are attributed to Sus scrofa, as comparedto 7232 for humans. The dominant protein was identifiedfrom each of the gel bands, although only four of thecomplete protein sequences of the 11 proteins identifiedwere present in the database for the pig and two morepresent in fragments. Thus, it is possible to identify suchproteins, provided there is a high degree of similaritybetween protein sequences of the species being studiedand protein sequences of the sequences in the database.When similarity is lower, protein identification is moredifficult and requires MS/MS sequencing data and soft-ware specifically designed to allow for amino acid sub-stitutions (23).

Protein Identification Strategies. The question ofhow definitive the identification is often arises afteranalysis of the data. In theory, a single complete peptideMS/MS fragmentation pattern of a peptide of 1 kDa isenough to define the protein or protein family, as can beseen in the HSP 90 MS/MS spectra in Figure 6. However,many of the peptides that are fragmented in the LC-MS/MS analysis do not give complete fragmentation series.This is almost inevitable in an automated setting, as theparameters for fragmentation cannot be optimized foreach peptide, and the peptide is often selected forfragmentation before or after it has reached its maximumelution from the HPLC column. If the peptide is large,there are usually gaps in the ion series, and the multiplycharged parent ions may have singly and multiplycharged fragment ions. In an ion trap system like theLCQ, the spectra recorded are not the full profile spectra,

and the charge state cannot be determined from the MSspectra unless a Zoom scan is also performed. The massaccuracy is also limited in full scan mode, so thatdifferences between certain amino acids, like K and Q,cannot be determined, and if the isolation is not restrictedto the monoisotopic peak, amino acids differing by 1 amucannot always be distinguished in the MS/MS spectra.There can also be fragments resulting from the neutralloss of water or ammonia from b or y ions that are notnecessarily included in the search program. Thus, theminimum number of peptides usually relied upon foridentification of a true unknown is three, of a minimumquality of 50-75% of the expected b and y ions beingseen, with a higher percentage of fragments ions neededfor low mass peptides and a greater number of ions forhigh mass peptides. One of the main concerns in an ESIrun occurs when no result is obtained, due to either poorin-gel digest or instrumentation difficulties. In oneexample, no identification is made for gel band 7 fromthe HPLC-ESI analysis, whereas the MALDI analysiscontains enough peptides for a clear identification.Because the ESI analyses were repeated at other times(19), there was no need to repeat this particular analysis.Repeating the analysis is a time-consuming process,requiring a minimum of an additional HPLC run ifsufficient sample remains for analysis and possiblyadditional sample workup. However, with the develop-ment of microspray HPLC at nanoliter flow rates, thesample quantity required is reduced. Alternatively, thetime required to collect data can be decreased by use ofnanospray with no HPLC separation of peptides (24, 25).

For MALDI spectra, a different set of parameters isnecessary to determine the likelihood of the match. TheMOWSE scoring system is in common use and is usefulfor sorting the output of the database search. However,the actual value of this number is not as important asthe gap between the score of the best hit and thesubsequent hits. The key parameters for determining thelikelihood that the protein identified is correct are asfollows: (i) number and percentage of peptides identified,(ii) intensity of ions of peptides matched, (iii) massaccuracy for peptides, and (iv) restrictions on the data-base search. In addition, information from the samplederived independently such as gel molecular mass range,pI, or species can be used for identification purposes butcan also be misleading. There can be no single parameterthat suffices for positive identification by peptide map-ping, but the set of parameters together can provide verystrong proof. For example, identification of a protein fromat least six peptides that correspond to the most intensepeaks in the mass spectrum and provide the best hit inan unrestricted database search when using a masstolerance of 75 ppm is generally a positive. Currentdelayed extraction reflectron MALDI instruments havea resolution of 7000 Da or better, which can give massesof 50 ppm or less using close external calibration. Newmethods for robotic spotting of targets and automateddata acquisition make close calibration possible on aroutine basis, and technology for plate calibration elimi-nates the need for repeated calibration. Such calibrationtechniques will make it feasible to utilize the highresolution of MALDI-TOF to generate accurate massmaps, reduce database search time, and increase theconfidence of results. These strategies work for proteinseither in the database or for those that show highsimilarity to database proteins, as the data show. Even


Figure 6. MS/MS identification of HSP 90 R and HSP 90 â. (A) The base peak MS chromatogram is shown for the HPLC-ESI-MS/MS run of gel band 4 containing HSP 90 R and â. Peptides eluted from the column are sent directly to the mass spectrometerand appear in the chromatogram between 7 and 34 min after the start of the HPLC run. The peaks are labeled with the m/z of themost intense ion eluting in each peak. The peptides whose MS/MS spectra are shown in B and C eluted at 28 and 29 min, respectively,as shown by boxes. (B) The MS/MS fragmentation spectra from the doubly charged ion at m/z 905, with the b and y ion assignmentsfrom the sequence of the HSP 90 â tryptic peptide, HSQFIGYPITLYLEK. (C) The MS/MS fragmentation spectra from the doublycharged ion at m/z 890, with the b and y ion assignments from the sequence of the HSP 90 R tryptic peptide, HSQFIGYPITLFVEK.The ions in boxes b12, b13, and y4 highlight the change in fragment ion masses due to the difference in sequence between the twopeptides.


for a species like minipig, which is poorly represented inthe database, the MALDI mass map successfully identi-fied the dominant protein(s) in each band. A closeexamination of the proteins identified shows that theseare all proteins that are highly conserved. When thesimilarity between species is lower, a peptide massmapping strategy is not feasible, as an average of just10% deviation between the amino acid sequences cantheoretically result in different masses for every trypticpeptide. While an unrestricted database search is themost thorough method of investigation, the search isoften restricted to limit the number of proteins matched.High molecular mass proteins are excluded because theymay have such a large number of possible digest peptidesthat false positive matches result. This is avoided byrestricting the database search when the protein isknown to be of lower molecular mass, as from gelsamples. In addition, when no definitive hit is made inan unrestricted search, the species can be limited to thegroup expected, such as mammals. However, restrictingthe species too narrowly may miss the correct identifica-tion of the protein, which may result from a contaminantoriginating from a species differing from the speciesunder investigation. Software developments for improvedscoring of peptide mass mapping continue and willeventually make automated discrimination between posi-tive and tentative identification of proteins possible (26).

In conclusion, we have found both MALDI-MS andHPLC-ESI-MS/MS to be effective tools for identifyingproteins separated on a SDS-PAGE gel. Both techniquesare capable of identifying two components in a singleband. They are both able to identify proteins from thesequences of a different species. MALDI-MS has theadvantage of high throughput and slightly higher aminoacid coverage. The single spectrum can be evaluated toassess the completeness of identification, and data stor-age space is minimal. HPLC-ESI-MS/MS can identify theprotein from fewer peptides and generates the sequencingdata necessary for similarity searching or cloning. Theuse of MALDI for initial protein identification followedby HPLC-ESI-MS/MS or nanospray analysis on previ-ously unidentified proteins has proved successful inefficiently identifying many proteins (13-17). The QTOFdetector design provides high mass accuracy for MS andMS/MS modes and can be employed with both MALDIand ESI (27). Alternatively, a MALDI-TOF/TOF designcan also deliver high sensitivity and mass accuracy (28).When fully automated, these advanced mass spectrom-eters will provide definitive protein identification dataand sequence tags for similarity searching.

While protein identification is achieved successfully byMALDI and ESI, typical coverage is in the range of 10-20%. The low coverage makes it difficult to find post-translational modifications, for the likelihood of detectinga specific peptide with 100% abundance is less than 1 in5. If the abundance is less, the likelihood drops further.Thus, alternative strategies are usually necessary whenlooking for modified peptides. MacCoss et al. (29) haveused a triple digest and two-dimensional LC-MS/MSapproach to increase protein coverage to 90% in order todetect modifications. Software developments such as theScoring ALgorithm for Spectral Analysis (30) or selectiveion monitoring (31) can help identify peptides with knownmodifications, while enrichment strategies can be em-ployed prior to MS analysis. For chemically inducedmodifications, a comparative MALDI-MS strategy fol-

lowed by targeted ESI-MS/MS has proved successful incharacterizing both known and novel modifications (32).These posttranslational modifications remain the mostimportant challenge for protein MS.

Acknowledgment. This work was supported in partby awards from the National Institute of General MedicalSciences to S.S.L. (GM 56321), the National Institute ofEnvironmental Health Sciences Center Grant (P30-ES07784), and Training Grant T32-ES07247 to K.M.T.MALDI spectra were acquired in the Mass SpectrometryFacility of the University of California at San Franciscounder the direction of Dr. Al Burlingame and supportedby NIH Grant NCRR RR01614.

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