epitope mapping of monoclonal antibodies by mass spectrometry: identification of protein antigens in...

Epitope Mapping of Monoclonal Antibodies by Mass Spectrometry: Identification of Protein Antigens in Complex Biological Systems

Lu Yu and Simon J. Gaskell Michael Barber Centre for Mass Spectrometry, Department of Chemistry, UMIST, Manchester, Unitecl Kingdom

Jayne L. Brookman Schoo~ of Biological Sciences, University of Manchester, Manchester, United Kingdom

We describe the application of immunoaffinity extraction and mass spectrometry to the analysis of Tyl Gag protein in lysates of Saccharomyces cerevisiae. A magnetic bead-conjugated monoclonal antibody was used to achieve selective extraction, the specificity of which was established by matrix-assisted laser desorption/ionization mass spectrometric (MS) analysis of an e~tract of the lysate of cells overexpressing the Tyl Gag protein. MS analysis of similar extracts of lysates following tryptic hydrolysis confirmed selective extraction of the epitope- containing peptide fragment. Sufficient sensitivity was achieved to allow the application of this approach to the analysis of lysates of wild-type cells. Furthermore, the sequence of the epitope-containing peptide was confirmed by electrospray-tandem MS. To our knowledge, this constitutes the first report of the application of immunoaffinity extraction and tandem MS analysis to the characterization of an antigen recovered from a complex cellular system. (J Am Soc Mass Spectrom 1998, 9, 208-215) © 1998 American Society for Mass Spectrometry

A common approach to the characterization of an organelle or structure within the cell is to raise antibodies against the structure and then deter-

mine the nature of the antigen recognized by the antibodies generated. Initially, for example, information on the size of a proteinaceous antigen may be determined by western blot analysis. Enzymatic treatment of the protein mixture may then be used to determine whether the antigen protein shows any post-transla- tional modifications, such as glycosylation or phosphor- ylation, and if so whether this alters the antibody binding. In order to identify more completely the protein with which the structure-specific antibody reacts, any antibodies raised may be used to screen appropri- ate cDNA expression libraries. This approach has been used with considerable success in the identification of structural proteins, e.g., cytocentrin in the centrosome [1]. However, this antibody screening method for cDNA libraries is of limited use where the antibody generated recognizes nonlinear or discontinuous epitopes or recognizes epitopes after secondary modifications (as described above), or where the target

Address reprint requests to Professor S. J. Gaskell, Michael Barber Centre for Mass Spectrometry, Department of Chemistry, UMIST, Manchester M60 IQD, L K. E-mail: [email protected]

protein is poorly represented in the cDNA library. An alternative approach which circumvents these prob- lems, as it does not rely on expression of the antigen, is to determine the epitope sequence recognized by the antibody. This sequence can then be used to search protein sequence databases for identical or homologous sequences. Furthermore, the sequence can be used to produce an oligonucleotide probe for direct screening of genomic or cDNA libraries.

Several antigen epitope analysis methodologies are available. The PEPSCAN technique (Cambridge Re- search Biochemicals, Cheshire, UK) uses enzyme-linked immunosorbent assays (ELISA) with sets of overlap- ping synthetic peptides spanning the known protein sequence. This approach has been used successfully for many different protein/antibody combinations [2-6] including the Tyl structural protein used in this report [7]. However, this is an expensive technique and is only really cost effectiive for characterizing a series of antibodies raised against a known protein. A similar approach suitable for both known and unknown antigens is the screening of peptide libraries. This method relies on recognition by the antibody of a linear synthetically produced peptide sequence and so is not suitable for detection of conformational or post-translationally modified epitopes.

~ 1998 American Society for Mass Spectrometry. Published by Elsevier Science Inc. 1044-0305/98/$19.00 PII $1044-0305(97)00250-X

Received July 22, 1997 Revised October 20, 1997

Accepted October 21, 1997

J A m Soc Mass Spect rom 1998, 9, 208-215 IDENTIFICATION OF PROTEIN ANTIGENS IN CELL LYSATES 209

An alternative approach to epitope analysis of monoclonal antibodies is used here. This methodology is based on an immunoaffinity step for isolation of the recognized antigen followed by mass spectrometric (MS) analysis. Several recent reports have described the application of MS to epitope mapping. Suckau et al. [8] compared the pattern of proteolytic degradation of a free peptide with an antibody-bound, and hence partially protected, peptide using plasma desorption MS. An equivalent approach has been used for an immobi- lized antibombesin antibody and its antigen, the gas- trin-releasing peptide [9]; in this instance (and in most other recent studies in this area) matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) MS analysis was used. Tomer and co-workers [10] recently reported the application of the same proteolytic footprinting/MALDI MS strategy for the characterization of the functional epitope on the HIV-1IIIB Gag protein. An alternative approach involves initial proteolytic digestion followed by immunoaffinity extraction of epitope-containing fragments. Zhao and Chait [11] followed this strategy for the screening of linear epitopes in the 26-amino acid peptide, melittin. Macht and co-workers [12] have pointed out that immobiliza- tion of the antibody may not be necessary for application of either the proteolytic footprinting or prior enzy- mic digestion approach when dealing with simple systems; in their work, separation of nonepitope peptides from antibody-bound peptides was achieved by ultrafiltration. A recent study by Vouros, Karger and coworkers [13] adopts an approach to screening of proteolytic fragments using affinity capillary electro- phoresis in combination with electrospray MS.

We describe here the development of an immunoaffinity-based mass spectrometric epitope analysis method for use in complex biological systems, such as whole cells, with identification of protein antigens at physiologically relevant levels. The reaction of the antigen and antibody takes place in solution directly from the cellular lysate. The system used for development of the method is the well-characterized Tyl retrotransposon present in the yeast Saccharomyces cerevisiae. The Tyl retrotransposon produces a virus-like particle, com- posed predominantly of a structural, Gag-like protein, during its replication cycle [14]. Many Tyl Gag con- structs including both C- and N-terminally truncated forms have been found to assemble when overex- pressed in yeast cells and have been used as a model system for architectural characterization of the Tyl virus-like particle [7, 15]. We have used a magnetic bead-conjugated monoclonal antibody that binds to a known epitope [7] to precipitate specifically the Tyl Gag or a fragment from whole and protease-digested yeast cell lysates. We have then characterized the affinity-extracted protein or peptide reacting with the monoclonal antibody by both MALDI-TOF and electrospray tandem MS. Furthermore, we have demonstrated the sensitivity of the system by successful extraction of the

antigen from yeast cell lysates containing only the wild-type, low levels of Tyl Gag protein.

Methods

Yeast Strains

Strains of Saccharomyces cerevisiae used were based on the host strain MC2 (cir+, leu2-3,112, ura3-5, trpl, pep4-3, prcl-407, prb1-1122) as described previously [7]. The strain OGS40 was made from transformation of MC2 with a 2 u*-based plasmid to overexpress the C-terminally truncated Tyl Gag protein, pl-381 (con- sisting of the first 381 amino acids of the Tyl Gag protein sequence plus an additional fiw~ amino acid residues, sequence AGSGK, introduced during plasmid construction). Cultures were grown in minimal yeast media, with galactose induction for the overexpressing cell lines [16].

Virus-like Particle Preparations

Purified MA5620 virus-like particles (VLPs) prepared from OGS40 yeast cells expressing the C-terminally truncated TYA protein, pl-381, were kindly provided by British Biotech plc (Oxford, UK). Samples were stored in small portions at -80°C. Monomeric pl-381 protein was prepared for MS analysis by dissociation of the particles with 0.5 M urea at 20°C. The urea was then removed from the solution by dialysis against 10 mM ammonium acetate, pH 6.5. The recovery of monomer from the Tyl VLPs was estimated to be 50%-80%.

Preparation of Antibody Beads

The anti-Tyl monoclonal BB2 [7] was purified by protein G affinity chromatography (Pharmacia, Uppsala, Sweden). The purified antibody was then coupled to magnetic microcellulose beads according to the manu- facturer's protocol (Scigen, Sittingbourne, Kent) using carbonyldiimidazole. The BB2-cellulose beads were then stored at 4°C in TEN-azide buffer (10 mM tris-HC1, pH 7.4; 2 mM EDTA; 140 mM NaC1; 0.05% NaN3).

Preparation of Yeast Cell Lysates

Yeast cell lysates were prepared for immunoaffinity extraction by vortexing cell pellets with glass beads in TEN buffer with a cocktail of protease inhibitors. Ly- sates were clarified by centrifugation (2000 x g, 5 min) before use. The protein concentration of cleared lysates was in the range 1.0-1.5 mg mL -1. Lysates were stored at -20°C when not analyzed immediate.ly. Trypsin- digested lysates were prepared by addition of 33 p,L of trypsin (2 mg mL-1 in TEN buffer; Sigma, UK) to 1 mL of cell lysate. The mixture was then incubated for 1.5 h at 37°C.

210 YU ET AL. J Am Soc Mass Spectrom 1998, 9, 208-215

SDS-PAGE and Western Blot Analysis of Tyl VLPs and Cell Lysates

Samples were electrophoresed on 10% SDS-PAGE gels and either visualized by Coomassie blue stain or blotted onto nitrocellulose using standard protocols [17, 18]. Tyl Gag proteins on nitrocellulose membranes were visualized by incubation with the BB2 antibody or another anti-Tyl antibody, TYG3 (reacting with the N- and C-terminal regions of the Tyl pl-381 protein, respectively [7]). Detection was achieved by labeling with peroxidaseqabeled rabbit antimouse antibody (DAKO, Buckinghamshire, UK) using 4-chloro-l-naph- thol (Sigma) as substrate in the presence of H20 2.

hnmunoaffinity Extraction and Elution

Tyl Gag proteins were removed from VLP solutions or cell lvsates by the addition of 20 IzL (equivalent to apprc, ximately 1 mg of beads) of BB2 beads. The mixtures were then incubated at 20°C for 1.5 h with gentle heating; the beads were subsequently removed from the solution by concentration with a rare earth magnetic bar (Scigen). The beads were washed with TEN+0.1% Tween-20 (3 × 0.5 mL), followed by water (HPLC grade, Rathburn Chemicals, Peeblesshire, Scotland; 5 × 0.5 mL) before elution of antigen. Samples for analysis by electrospray MS were given extra washing steps (3 × 0.2 mL PBS) before the water washes to remove detergent which may interfere with the MS analysis. Anti- gens specifically bound to the BB2 beads were then eluted with an aqueous solution of glycine HCI (5 txL, 0.1 M, pH 2.3) immediately before MS analysis. After elution of bound material, the beads were washed (3 × TEN l~uffer) and stored in TEN azide until reuse.

MALDI-TOF Mass Spectrometry

Samples were prepared for MS by mixing 2 p~L of the BB2 beads eluate with an equal volume of matrix solution. Sinapinic acid was used as matrix for the analy:sis of the intact Tyl protein samples and c~-cyano- 4-hydroxycinnamic acid for the trypfic digests; both matrices were dissolved in water:acetonitrile (2:1) containing 0.1'14, trifluoroacetic acid. A portion of the sample-matrix mixture (2 /xL) was spotted onto the target for analysis. Mass spectra were obtained using a Tof- Spec instrument (Micromass UK, Manchester, UK) equipped with a 337 nm nitrogen laser. The accelerating voltage was 25 kV in linear mode and 23.6 kV in reflectron mode; the signal digitization rates were 250 and 500 MHz, respectively. Data from 50 laser shots were averaged for each spectrum using Micromass Opus Motif software. External mass calibration in linear mode was performed using bovine serum albumin samples in sinapinic acid matrix. Calibration in reflectron mode used a mixture of gramicidin S and bovine insulin in c~-cyano-4-hydroxycinnamic acid matrix.

EIectrospray Mass Spectrometry

Electrospray mass spectra were obtained using a Micro- mass Quattro tandem quadrupole mass spectrometer, upgraded to Quattro II specifications. The samples eluted from BB2 beads were desalted chromatographi- cally by application to a reusable reversed-phase cartridge (1 × 15 ram; Peptide Trap, Michrom BioRe- sources, Pleasanton, CA). The cartridge was washed with 300/~L aqueous formic acid (0.1%) before recovery of the protein or peptide by elution with 80 p,L of C H 3 C N / H 2 0 (80/20) containing 0.1% formic acid. Por- tions of the eluate were infused into lhe electrospray source at a flow rate of 3 /~L/min using a syringe driver (Harvard Apparatus, South Natick, MA). For conven- tinnal MS analyses, the resolution was set to achieve peak widths of -~:1 m/ z unit for singly charged ions. For tandem MS the resolution of the first quadrupole was set to achieve peak widths of singly charged precursor ions of "~2 m / z units; the equivalent figure for product ion analysis was 1.2-1.8 m / z units. The mass spectrometer was repetitively scanned at a rate of 100 m/z units/s. Conventional MS data were accumulated for 1.5 min and tandem MS data for either 8 or 18 min. The collision energy used in the tandem MS analyses was 30-45 eV. Argon was used as collision gas at a recorded manifold pressure of 1.3 × 10 -:~ mbar.

Results

Mass Spectrometric Analysis of the 7 y l Structural Proteiu from Virus-like Particle,;

MA5620 virus-like particles purified from yeast cells overexpressing file truncated Gag protein pl-381 were analyzed by MALDI-TOF MS. These particles are com- posed of several hundred copies of the monomeric pl-381 protein [15] and so require dissociation before analysis. The MALDI-TOF mass spectrum obtained for the dissociated MA5620 VLPs (not shown) included a prominent signalt at m/z 4;{,224, attributable to the singly protonated protein. Doublv and triply charged forms were also present, together with an intense signal associated with the protonated dimer. The derived estimate of molecular mass, 43,223, is within 0.5% of the (M+H) + mass of 43,002 calculated from the average mass of the amino acid sequence (taking into account the additional five amino acid sequence introduced during plasmid construction [19]. Figure 1 provides further evidence for the identity of the Tyl pl-381 protein based on SDS-PAGE and western blot analyses with two different anti-Tyl monoclonal antibodies. (The discrepancy between the molecular weight estimate based on gel mobilitv and that obtained by MALDI MS illustates the common inaccuracy of gel- based determinations.)

J Am Soc Mass Spectrom 1998, 9, 208-215 IDENTIFICATION OF PROTEIN ANTIGENS IN CELL LYSATES 211

1 2 3 4

B C

A Figure 1. SDS-PAGE and western blot analyses of samples prepared for mass spectrometry. Panel A shows a Coomassie blue-stained gel with the following components: Lane 1: molecular weight markers; lane 2: the purified Tyl protein isolated from Tyl virus-like particles and used for mass spectrometric analysis. The spot corresponding to Ty protein appears between molecular weight markers of 45 and 66 kDa. Lanes 3 and 4: }:east cell lysates prepared from cells overexpressing the Tyl pl-381 structural protein, before (lane 3) and after (lane 4) extraction with antibody beads. Panels B and C show western blots prepared using antibodies recognizing either an N-terminal (panel B) or a C-terminal section (panel C) of the Tyl structural protein. Lane 1: purified Tyl protein (as panel A, lane 2); lane 2: extract of yeast cell lysate; lane 3: yeast cell lysate (as panel A, lane 3).

Optimization of Immunoaffinity Extraction Procedures

The extraction procedure developed for cell lysate in- cubations with antibody beads (for subsequent MS analysis) was based on conditions used for similar immunoaffinity procedures such as western blotting and ELISAs. The following parameters were evaluated for optimization of extraction and MS: (a) conditions for the washing of the antibody beads after initial extraction of lysates, (b) choice of elution agent for recovery of bound protein, and (c) the matrix used for MALDI-TOF MS. The removal of detergent used during initial washing of the antibody-beads/antigen complex (as described in the Methods section) was found to be particularly important in order to avoid interfering signals during MS analysis. Aqueous glycine hydrochloride achieved efficient dissociation of the antigen from the antibody bead-antigen complex and did not interfere with the subsequent MS analysis [9]. Aqueous trifluoroacetic acid (1%) was also effective for dissociation of the complex but was less useful in the subsequent MALDI-MS analysis because matrix crystal formation was impaired, presumably by material co-extracted from the antibody-coupled beads.

'oo 1

>= %

nr"

[M+2H] 2" 21446

{M+H]" 4 2 9 6 9

q

0 t - - - - . . ~ - w ~ . 10000 20000 30000 40000 50000

Figure 2. MALDI-TOF mass spectrum of Tyl structural protein isolated from a lysate of ),east cells (strain OGS40) overexpressing the Tyl pl-381 structural protein.

Analysis of Tyl Protein Extracted frorn Yeast Cell Lysates

Lysates were prepared from cells (OGS40 strain) overexpressing the Tyl pl-381 protein and then incubated with magnetic beads coupled to the anti-Tyl monoclo- hal antibody BB2. The MALDI-TOF mass spectrum of the protein sample eluted from the BB2 beads is shown in Figure 2. The major peak present at m/z 42,969 shows good agreement with the calculated molecular mass of 43,002 for the Tyl p1-381 construct. A peak at m/z 21,446 is attributed to the doubly protonated form. The BB2 beads are therefore successfully extracting from the complex yeast cell lysate a single protein with a spectrum equivalent to that obtained from the dissociated Tyl virus-like particle preparations. The identity of the eluted protein as Tyl pl-381 protein was supported by western blot analysis using both a C-terminal-reacting and an N-terminal-reacting anti-Tyl antibody (Figure 1B,C). No other protein spots were revealed by western blotting with either antibody. Figure 1 also shows the results of Coomassie-blue staining of SDS-PAGE separations of the OGS40 cell lysate before and after extraction with the antibody beads. In neither case is a discrete spot observed corresponding to the Tyl protein and the gross protein composition is not altered by extraction with the antibody beads. (Compare lanes 3 and 4 in Figure 1A.) Determinations of the concentration of the Tyl protein in the cell lysates are necessarily very approximate; based on western blotting and densitometry, however, it is estimated that Tyl represents a maximum of 2% of total protein in the overexpressing yeast strain. Thus, 1 mL of lysate, corresponding to 1--1.5 mg total protein, is expected to contain 0.5-0.7 nmo} of Tyl protein. West- ern blotting (not shown) of a cell lysate after extraction with antibody beads revealed that extraction of the Tyl protein was incomplete so an estimate of 0.5-0.7 nmol Tyl in an extract of 1 mL of lysate represents an upper limit.

212 YU ET AL. J Am Soc Mass Spectrom 1998, 9, 208-215

<E

r r

A

B

1 0 0

%

T 2

+ 1640.4

i t

T2+3 2360.1 'l+,

1640.3

o 1 ~ 2000

T2+3+4 2747.4

~ , L d . . . . . t . L . L ~ . J . . . . . . . . . . . . . . . . . . . J . . - - I. . t . d ,

3000 " 4(JO0 ' 5000 . . . . . . m / z

1 0 0

%

T 2 1639.9

b . , i ~ 06t,i,,.4CdNmJl~ ........ L .....................

+ ~ 2 ~ ~ 4 ~ ~ ~ z

Figure 3. MALDI-TOF mass spectra of samples recovered by immunoaffinity extraction of yeast cell lysates, after proteolytic digestion with trypsin. A: Spectrum of the extract of 1 mL of digested lysate of the cells overexpressing Tyl pl-381 protein. T2 denotes the second expected tryptic fragment. B: Spectrum of the extract of a mixture comprising 0.1 mL tryptic lysate from the cells overexpressing the Tyl pl-381 protein together with 0.9 mL undigested, normal yeast cell lysate. C: Spectrum of the extract of I mL of digested lysate of cells expressing Tyl protein at wild-type levels.

Extraction and Analysis of Protein Fragments from Trypsin-digested Cell Lysates

An enzymatic digest was made of the whole cell lysate before extraction with the antibody beads. Figure 3A shows the mass spectrum of the sample recovered by immunoaffinity extraction of a tryptic digest of a lysate from yeast cells (OGS40) overexpressing the Tyl pl-381 protein. The spectrum shows a single peak at m/z 1640.4 which is consistent with a singly protonated ion deriw_~d from the predicted tryptic fragment (T2) of Tyl pl-381. Analyses of replicate (n = 4) cell lysates on separate occasions indicated a satisfactory reproducibility of determination of the peptide mass: 1639.7 _+ 1.1 Da (mean + S.D.). This fragment incorporates residues 25-39 (EVHTNQDPLDVSASK) and thus encompasses the known epitope of the BB2 antibody at residues 27-32, as determined by PEPSCAN analysis [7].

The sensitivity of the immunoaffinity extraction and mass spectrometric analysis procedure was tested by dilution of the trypsin-digested lysate of overexpressing cells (OGS40 strain) with lysate from the nonoverexpressing host yeast strain, MC2. Figure 3B shows the analysis of a sample extracted from a mixture of 0.9 mL of MC2 lysate and 0.1 mL of the digested OGS40 lysate (compared with 1 mL of OGS40 lysate used to obtain the data shown in Figure 3A).

It is apparent from Figure 3B that the trypsin present in the OGS40 lysate reaction mixture has partially digested the Tyl protein present at wild-type levels in the MC2 yeast lysate. The spectrum shows major peaks

for the T(2+3) tryptic fragment (residues 25-45) at m/z 2360.1 (calculated value 2360.5) and the limit digest fragment T2 at m/z 1640.3. A minor signal corresponds to the T(2+3+4) tryptic fragment (residues 25-49) at m/z 2747.4 (calculated value 2748.0). The partially digested forms were subject to complete digestion by increasing the incubation time after addition of the wild-type lysate or by adding further trypsin; subsequent MALDI-TOF MS analyses gave spectra identical to that shown in Figure 3A.

Extraction of Wild-type Yeast Cell Lysates

The dilution experiments described above suggested that the immunoaffinity extraction-MALDI MS technique was sufficiently sensitive to allow characterization of the Tyl epitope at wild-type levels. Tryptic digests of nonoverexpressing MC2 lysates were therefore analyzed for confirmation of the system sensitivity. The reproducibility and robustness of the procedure were also established by analyzing several isolates, by extracting portions of the same isolate after varying times of storage and by assessing the utility of antibody beads for multiple re-use.

Figure 3C shows the MALDI-TOF mass spectrum of the immunoaffinity extract of a digest of an MC2 lysate. (As with the analyses of the OGS40 strain, 1 mL of lysate was used, corresponding to an approximate cell number of 5 × 108 and an amount of protein of 1.0-1.5 mg; a 20% portion of the extract was used for MS

J Am Soc Mass Spectrorn 1998, 9, 208-215 IDENTIFICATION OF PROTEIN ANTIGENS IN CELL LYSATES 213

analysis.) The spectrum shows a single peak of m/z 1640.3. The concentration of Tyl protein in the wild- type MC2 lysate is estimated by western blotting and ELISA assay to be at least an order of magnitude lower than in the overexpressing OGS40 strain.

Identical MALDI-TOF mass spectra showing single components were obtained from analyses of several cell lysates. Analyses of aliquots of the same cell lysate on four separate days confirmed the reproducibility of determination of the mass of the extracted peptide: 1638.4 + 0.4 Da (mean +_ S.D.). The beads were found to be reusable after elution of antigens with consistent data observed for at least five consecutive uses. The volume of lysate extracted could be reduced to 0.5 mL without significant loss in signal quality (data not shown).

r r -

A

1 0 0 - . . . . . . . x l 0 . . . . . . .

y , Y= ys

[l~ ~ Y3 b4 Yo

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Yt=

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. . . . . . . , . . . . , - , . . . . , . . . . . . m / z

8 0 0 1 0 0 0 1 2 0 0

p y|=. 100

Electrospray Analysis The further development of the affinity extraction-MS approach described above relies on the sequence determination of tryptic or other fragments eluted from antibody beads. The searching of protein databases for location of the protein epitope recognized by mass spectrometric analysis will be greatly facilitated if molecular weight information is supplemented by partial or total sequence data [20, 21]. Supplementary experiments were therefore performed to establish whether the sample preparation techniques described above were compatible also with electrospray tandem MS analysis to provide sequence data.

The mass spectrum obtained by conventional electrospray MS analysis of an immunoaffinity extract of the tryptic hydrolysate of a wild-type cell lysate (not shown) clearly indicated the compatibility of the extraction procedure with electrospray. Two prominent ions were observed, attributable to [M+2H] 2~ and [M+3H] 3+ ions derived from a single peptide. Analyses of replicate extracts of a wild-type cell lysate yielded a peptide mass of 1638.9 + 0.3 Da (mean -+ S.D.; n = 9), in good agreement with the estimates of molecular mass from MALDI MS analysis.

The prominence of the triply protonated ion in the conventional ESI spectrum of a tryptic peptide is consistent with the presence of a histidine, providing a third basic site in addition to the N-terminal amine and the side chain of the C-terminal residue (lysine or arginine). The sequence of the extracted peptide was established by ESI-tandem MS using both [M+2H] 2. and [M+3H] 3. as precursor ions. The respective product ion spectra are shown in Figure 4A and 4B; the assignments of product ions are shown in Figure 5. (The nomenclature used is the Biemann variant [22] of the original Roepstorff and Fohlman proposal [23].) Both spectra include ions of m/z 147 attributable to the y~ ion from C-terminal lysine (and lack a significant ion of m/z 175 which would indicate C-terminal arginine), providing a suitable starting point for facile interpreta- tion of the spectra, y-Series ions associated with cleav-

¢:

<~,

r r

B

% Y6

L D3 ÷

L,JI, aK, / , , . C - ' . . • . . . l - . . J I I t . h i l l ,) , L ~ m / z

2 0 0 460 660 ' 860

Figure 4. Tandem MS analyses of the extract of 1 mL of digested lysate of cells expressing Tyl protein at wild- type levels. A: Product ion spect rum of the [M+2H] :~- ions derived from the T2 tryptic fragment. B: Product ion spectrum of the [M + 3H] 3. ions derived from the T2 tryptic fragment.

age of 12 of the 14 peptide bonds are observed in one or both of the product ion spectra.

D i s c u s s i o n

Antibodies against known antigens have long been used for the recovery prior to MS analysis of antigens such as steroids in blood plasma [24, 25] and eico- sanoids in urine [26, 27]. The development of the electrospray and MALDI methods have made the immunoaffinity extraction/MS combination attractive for the selective analysis of more polar and higher molecular weight compounds. Initial applications of this approach to the analysis of peptides and proteins present in solutions of simple mixtures [8, 9, 11, 28-30] have been extended recently to the analysis of more complex biological systems [31-37]. In the present work, extraction of yeast cell lysates (and tryptic digests thereof) with a monoclonal antibody coupled to magnetic beads has been shown to provide samples suitable for analysis by both MALDI and electrospray MS. The test system used for the development of these combined immunoaffinity extraction/MS methodologies, the Gag protein of the Tyl retrotransposon, has involved a

214 YU ET AL. J Am Soc Mass Spectrom 1998, 9, 208 215

A

YI,3 [~,..Yll Y9 L,,.. Y5 L,,.- Y3L,.- Yl Y12 Yo Y4 Y2

B

b7 b I b3 b6

E~V-H-J-T-N-Q--"-ID L D V S - A S K DtyP~T D ~ ~ . ~

Y8 Y6YSY4 Y2Yl

Figure 5. Product ions from CAD of [M+2H] 2÷ ions (A) and [M+3H] 3. ions (B) derived from the T2 tryptic fragment, observed during electrospray tandem MS analysis of the extract of 1 mL of c, igested lysate of cells expressing Tyl protein at wild-type levels (]~igure 4).

well-characterized antigen-antibody combination. The methods described are quite rapid and, owing to the instrumental sensitivity, extremely sparing in terms of the biological material required. Samples prepared from extraction of lysates equivalent to 108 yeast cells were sufficient for five separate MALDI-MS analyses or a single electrospray tandem MS analysis. As empha- sized above, determination of the proportion of total protein accounted for by the Tyl Gag protein is necessarily very approximate; crude estimates suggest a figure of less than 2% in the overexpressing cells and substantially less in wild-type yeast. This suggests that further development of these procedures using antibodies raised against, for example, structural components within the cell need not be restricted to the most prevalent proteins. The avidity of the antibody for its substrate will, of course, limit the ultimate sensitivity of the assay for any individual antigen. Available evidence (based on gel mobilities before and after subjection to reducing conditions) suggests that the native structure of the Tyl Gag protein does not incorporate disulfide bonds Further evaluation is required to establish the applicability of the methods described here to the analysis of proteins amenable to proteolytic digestion only after reduction and alkylation.

The use of magnetic beads, rather than the Sepharose beads often used for other immunoaffinity extraction protocols, was particularly advantageous for extraction of the yeast cell lysates. The magnetic beads, together with the antigen-antibody complex, were concentrated using a magnetic bar, thus avoiding the contamination of the recovered solid phase by cellular debris that may result from centrifugation. Furthermore, the use of magnetic beads places fewer constraints on the sample volumes used for extraction or washing. The selectivity of reco~ery of targeted cellular components will depend upon the specificity of the antibody used for extraction, as is ailways the case with immunoaffinity-based methodologies, such as western blotting, ELISA, immuno-

precipitation, and library screening with antibodies. With the present technique, however, a very high specificity of characterization is achieved by the MS and tandem MS analyses of the extract.

The use of tryptic fragment masses for the recognition of proteins on the basis of mass spectrometric data and database searching was introduced several years ago [38-42] and has been recently reviewed [43]. A number of refinements to the search routine may be incorporated, including the specification of full or partial sequence data for selected proteolytic peptides [20, 21]. The present work has demonstrated the facility with which extensive sequence data may be obtained by tandem MS analysis of an epitope-containing fragment recovered by immunoaffinity extraction. When the objective of the immunoaffinity extraction/MS analysis is identification of file cellular protein recognized by the antibody, rather than the sequencing of the epitope, the intact protein may be recovered (as demonstrated here) prior to proteolysis, MS characterization of the fragments, and database searching. Such an approach has recently been described by McCormack et al. [37]. As a complement to this strategy, contiguous amino acid sequences can be used to prepare oligonucleotide probes for polymerase chain reaction (PCR) amplifica- tion of the sequence from genomic DNA or mRNA. Fragments generated can then be used for library screening using DNA hybridization or PCR ap- proaches. Thus, even if the antibody recognizes a completely novel antigen, the sequence of which is not available on any database, tools can be made which make isolation and subsequent identification of the antigen possible.

This report has focused on the development of immunoaffinity extraction and MS for the confirmation of a linear cellular epitope incorporating no post-trans- lational modifications. The use of MS for the analysis of extracted proteins and of proteolytic fragments incorporating the epitope sequence should mean that the characterization of post-translationally modified epitopes can be achieved using the same approach. The applicability of these techniques to the determination of cellular components incorporating conformational (discontinuous) epitopes remains to be established. Proteo- lytic digestion of the antibody-bound protein antigen (that is, after immunoaffinity extraction of the cell lysate) may achieve this objective [10] but further work is required.

Acknowledgments This work was supported in part by a grant (GR/K18658) to SJG from the UK Engineering and Physical Sciences Research Council. LY is the recipient of an Overseas Research Studentship from the Committee of Vice-chancellors and Principals of the Universities of the UK. We are grateful to Micromass UK for generously providing access to the time-of-flight mass spectrometer.

J Am Soc Mass Spectrom 1998, 9, 208 -215 IDENTIFICATION OF PROTEIN ANTIGENS IN CELL LYSATES 215

References

1. Paul, E. C. A.; Quaroni, A. J. Cell Sci. 1993, 106, 967-981. 2. Gombert, F. 04 Werz, W.; Schluter, M.; Bayer, A.; Werner,

R. G.; Berthold, W.; Jung, G. Biol. Chem. Hoppe-Seyler 1994, 375, 471-480.

3. Tosser, G.; Delaunay, T.; Kohli, E.; Grosclaude, J.; Pothier, P.; Cohen, J. Virology 1994, 204, 8-16.

4. Schotanus, K.; Holtkamp, G. M.; Meloen, R. H.; Puijk, W. C.; Berkenbosch, F.; Tilders, F. J. H. Endocrinolo~ d 1995, 136, 332-339.

5. Harris, D. P.; Vordermeier, H. M.; Arya, A.; Mnreno, C.; Ivanyi, J. hnmunology 1995, 84, 555-561.

6. Harris, D. P.; Vordermeier, H. M.; Singh, M.; Moreno, C.; Jurcevic, S.; Ivanyi, J. Eur. ]. lmmunol. 1995, 25, 3173-3179.

7. Brookman, J. L.; Stott, A. J.; Cheeseman, P. J.; Burns, P. J.; Adams, S. E.; Kingsman, A. J.; Gull, K. Virology 1995, 207, 59-67.

8. Suckau, D.; KOhl, J.; Karwath, G.; Schneider, K.; Casaretto, M.; Bitter-Suermann, D.; Przybylski, M. Proc. Natl. Acad. SoL USA 1990, 87, 9848-9852.

9. Papac, D. I.; Hoyes, J.; Tomer, K. B. Protein Sci. 1994, 3, 1485-1492.

10. Parker, C. E.; Papac, D. I.; Trojak, S. K.; Tomer, K. B. J. lmmunol. 1996, 157, 198-206.

11. Zhao, Y.; ChaiL B. T. Anal. Chem. 1994, 66, 3723-3726. 12. Macht, M.; Fiedler, W.; Kurzinger, K.; Przybylski, M. Biochem-

i s t~ 1996, 35, 15633-15639. 13. Lyubarskaya, Y. V.; Dunayevskiy, Y. M.; Vouros, P.; Karger,

B. L. Anal. Chem. 1997, 69, 3008 -3014. 14. Sandmeyer, S. B. Curr. Opin. Genet. Dev. 1992, 2, 705-711. 15. Burns, N. R.; Saibil, H. R.; White, N. S.; Pardon, J. F.; Timmins,

P. A.; Richardson, S. M. H.; Richards, B. M.; Adams, S. E.; Kingsman, S. M.; Kingsman, A. J. EMBOJ. 1992, 11, 1155-1164.

16. Brookman, J. L.; Stott, A. J.; Cheeseman, P. J.; Adamson, C. S.; Hohnes, D.; Cole, J.; Burns, N. R. ViroloK V 1995, 212, 69-76.

17. Towbin, H.; Staehelin, T.; Gordon, J. Proc. Natl. Acad. Sci. USA 1979, 76, 4350-4354.

18. Laemmli, U. K. Nature 1970, 227, 680-685. 19. Mellor, J.; Fulton, S. M.; Dobson, M. J.; Wilson, W.; Kingsman,

S. M.; Kingsman, A. J. Nature 1985, 313, 243-246. 20. Mann, M.; Wilm, M. Anal. Chem. 1994, 66, 41390-4399. 21. Wilm, M.; Shevchenko, A.; Houthaeve, T.; Breit, S.; Schweig-

erer, L.; Fotsis, T.; Mann, M. Nature 1996, 379, 466-469.

22. Biemann, K. Biomed. Environ. Mass Spectrom. 1988, 16, 99-1111. 23. Roepstorff, P.; Fohlman, J. Biomed. Mass Spectrom. 1984, 11,601. 24. Gaskell, S. J.; Brownsey, B. G. CJin. Chem. 1983, 29, 677-680. 25. Gaskell, S. J. Biomed. Environ. Mass Spectrom. 1988, 15, 99-104. 26. Chiabrando, C.; Benigni, A.; Piccmelli, A.; Carminati, C.; Cozi,

E.; Rernuzzi, G.; Fanelli, R. Anal. Biochem. 1987, 163, 255--262. 27. Hubbard, H. L.; Eller, T. D.; Mais, D. E.; Halushka, P. V.;

Baker, R. H.; Blair, I. A.; Vrbanac, J. J.; Knapp, D. R. Prosta glandins 1987, 33, 149-159.

28. Hutchens, T. W.; Yip, T.-T. Rapid Commun. Mass Spectrom. 1993, 7, 576-580.

29. Hsieh, Y. L. F.; Wang, H. Q.; Elicone, C.; Mark, J.; Martin, S. A.; Regnier, F. Anal. Chenl. 1996, 68, 455-462.

30. Zhao, Y. M.; Muir, T. W.; Kent, S. B. H.; Tischer, E.; Scardina, J. M.; Chait, B. T. Proc. Natl. Acid. Sci. USA 1996, 93, 4020- 4024.

31. Nakanishi, T.; Okamoto, N.; Tanaka, K.; Shimizu, A. Biol. Mass Spectrom. 1994, 23, 230-233.

32. Nelson, R. W.; Krone, J. R.; Bieber, A. L.; Williams, P. Anal. Chem. 1995, 67, 1153-1158.

33. Nakanishi, T.; Shimizu, A.; Okamoto, N.; Ingendoh, A.; Kanai, M. ]. Am. Soc. Mass Spectrom. 1995, 6, 854-859.

34. Kishikawa, M.; Nakanishi, T.; Miyazaki, A.; Shimizu, A.; Nakazato, M.; Kangawa, K.; Matsuo, H. J. Ma~s Spectrom. 1996, 31, 112-114.

35. Wang, R.; Sweeney, D.; Gandy, S. E.; Sisodia, S. S. J. Biol. Chem. 1996, 271, 31894-31902.

36. Liu, C. L.; Bowers, L. D. J. Chromato~r. B Biomed Appl. 1996, 687, 213-220.

37. McCormack, A. L.; Schieltz, D. M.; Goode, B.; Yang, S.; Barnes, G.; Drubin, D.; Yates, J. R. Anal. Chem. 1997, 69, 767 776.

38. Mann, M.; Hojrup, P.; Roepstorff. P. Biol. Mass Spectrom. 1993, 22, 338-345.

39. Henzel, W. J.; Billeci, T. M.; Stults, J. T.; Wong, S. C.; Grimley, C.; Watanabe, C. Proc. Natl. Acad. Sci. USA 1993, 90, 5011-5015.

40. James, P.; Quadroni, M.; Carafoli, E.; Gonnet, G. Protein Sci. 1994, 3, 1347-1350.

41. Pappin, D. J. C.; Hojrup, P.; Bteasby, A. J. Ci~rr. Biol. 1993, 3, 327-332.

42. Yates, J. R.; Speicher, S.; Griffin, P. R.; Hunkapiller, T. Anal. Biochem. 1993, 214, 397-408.

43. Burlingame, A. L.; Boyd, R. K.; Gaskell, S. J. Anal. Chem. 1996, 68, 599R-651R.

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