Selective Detection of Membrane Proteins Without Antibodies
A Mass Spectrometric Version of the Western Blot*
David Arnott
,
,
Adrianne Kishiyama
,
Elizabeth A. Luis
,
Sarah G. Ludlum
,
James C. Marsters, Jr.¶ and
John T. Stults
Department of Protein Chemistry, Genentech, Inc., South San Francisco, California 94080
¶ Department of BioOrganic Chemistry, Genentech, Inc., South San Francisco, California 94080
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ABSTRACT
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A method has been developed, called the mass western experiment in analogy to the Western blot, to detect the presence of specific proteins in complex mixtures without the need for antibodies. Proteins are identified with high sensitivity and selectivity, and their abundances are compared between samples. Membrane protein extracts were labeled with custom isotope-coded affinity tag reagents and digested, and the labeled peptides were analyzed by liquid chromatography-tandem mass spectrometry. Ions corresponding to anticipated tryptic peptides from the proteins of interest were continuously subjected to collision-induced dissociation in an ion trap mass spectrometer; heavy and light isotope-coded affinity tag-labeled peptides were simultaneously trapped and fragmented accomplishing identification and quantitation in a single mass spectrum. This application of ion trap selective reaction monitoring maximizes sensitivity, enabling analysis of peptides that would otherwise go undetected. The cell surface proteins prostate stem cell antigen (PSCA) and ErbB2 were detected in prostate and breast tumor cell lines in which they are expressed in known abundances spanning orders of magnitude.
Tools for the measurement and analysis of gene and protein expression patterns are at the core of several recently defined disciplines, functional genomics, transcriptomics, proteomics, and subfields such as pharmacogenomics and pharmacoproteomics. Among these tools, differential display of mRNA is performed routinely using cDNA microarrays (1, 2). Fluorescence detection, together with the amplification of DNA using the polymerase chain reaction, allows such experiments to be performed with exquisite sensitivity, and the parallel detection of thousands of gene products enables high throughput measurements. For protein measurement, 2D1 PAGE is capable of resolving 2500 or more distinct protein spots (3), making it the highest resolution protein separation experiment yet devised. This venerable technique has undergone a rebirth because of advances in reproducibility and automation and the ability to identify most detectable proteins using mass spectrometry and sequence data base searching (4, 5).
More recently, a particularly powerful technique to emerge is the combination of liquid chromatography and mass spectrometry (LC-MS) or tandem mass spectrometry (LC-MS/MS). Because spectra from only a few peptides (or even a single peptide) can be sufficient to identify a protein, multiple components of a protein mixture can be identified (6). Several groups have used this technology to identify hundreds of proteins from the tryptic digests of crude cellular extracts (712). The isotope-coded affinity tag methodology (ICAT) first described by Gygi and colleagues (13, 14) has extended such experiments to allow relative quantitation of proteins between two samples. This technique involves differential labeling of proteins in two samples with affinity (e.g. biotinylation) reagents differing slightly in mass. After mixing and digestion of the samples the labeled peptides are isolated by affinity chromatography and analyzed by mass spectrometry. Each peptide is detected as two peaks in an LC-MS experiment. Tandem MS is used to identify the protein from which each peptide is derived, and the relative abundances of corresponding peaks reflect the amounts of protein in each sample from which they were derived.
As powerful and complementary as current genomic and proteomic tools are, they nevertheless suffer several shortcomings. Although tools such as cDNA microarrays are extraordinarily powerful for the simultaneous detection of thousands of gene products, mRNA levels do not necessarily correlate with protein expression levels (15, 16). 2D PAGE is of limited use in verifying DNA microarray results, because it is difficult to predict in advance which of the potentially thousands of spots corresponds to a given protein because of the spectrum of possible post-translational modifications. Alternatively, fluorescence-activated cell sorting, immunohistochemistry, Western blots, enzyme-linked immunosorbent assays, and other antibody-based approaches can be used to explore the expression patterns and biological function of proteins. These powerful, but often time-consuming, techniques are currently the methods of choice to expand on the results of mRNA-based experiments. Reliance on antibodies, however, makes this difficult to do quickly, because antibodies must first be generated for each target protein. Furthermore, an antibody that binds a native protein (as in immunoprecipitation) may not be useful for detecting the denatured protein on a Western blot. Thus, a technique is needed that is similar to a Western blot but does not require an antibody to each protein of interest. Such a technique should be rapid, sensitive, quantitative, and capable of identifying a specific protein out of extremely complex mixtures without bias or need for extensive purification of intact proteins.
We have developed an analytical procedure with the potential to meet these requirements based on the ICAT methodology of Gygi et al. (13). This experiment, dubbed the mass western in analogy to the Western blot, was applied to the detection of proteins from plasma membrane preparations of human cells without electrophoresis or other initial purification steps. Proteins assayed included the prostate stem cell antigen (PSCA) and the receptor tyrosine kinase ErbB2, which are over-expressed in significant numbers of prostate tumor and breast tumors, respectively.
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EXPERIMENTAL PROCEDURES
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Purification of Membrane Proteins
Breast tumor cell lines SK-BR-3 and MCF-7, or prostate tumor cell line PC3 transfected with the full-length sequence of PSCA, were used in these experiments. Eighty million of the transfected PC3 cells (designated clone 11) were used in the PSCA experiment. Fifty million MCF-7 cells and approximately half as many SK-BR-3 cells, normalized by total protein content, were used in the ErbB2 study. Cells cultured in flasks (175 cm2; Falcon) were harvested and disrupted with a Dounce homogenizer (17). In the ErbB2 experiment the cell lysate was centrifuged at 1000 x g for 5 min to remove intact cells and nuclei. The resulting post-nuclear supernatant was centrifuged at 16,000 x g for 15 min to produce a crude membrane pellet. In the PC-3 experiment the post-nuclear supernatant was layered over a 35% (w/v) sucrose solution and centrifuged in a SW55Ti rotor at 38,000 x g for 45 min. The membrane layer was collected, pelleted, and washed successively for 30 min with each of the following: 1) ice-cold 25 m
M Na2CO3, pH 11, to remove cytoskeletal proteins (18), 2) 0.5% Tween 20 (Calbiochem) at 4 °C, and 3) cold phosphate-buffered saline, with each step followed by centrifugation at 16,000 x g for 15 min. Protein concentrations were determined by bicinchonic acid assay.
d3 ICAT Reagent Synthesis
-N-iodoacetyl-
-N-biotinyl-
L-lysine trideuteromethyl ester was prepared in a two-step synthesis.
-N-biotinyl-L-Lysine (biocytin, 100 mg; Sigma) was suspended in methanol-d4 (2 ml; Cambridge Isotope Laboratory) and 4 N HCl/dioxane (2 ml; Pierce) in a sealed flask overnight, evaporated to dryness, and evaporated twice more from toluene. The resulting trideuteromethyl ester was used without further purification. The residue was dissolved in N,N-dimethylformamide (1 ml) and N,N-diisopropylethylamine (0.2 ml; Aldrich) and treated with iodoacetic anhydride (114 mg; Aldrich). After 30 min, the reaction mixture was loaded directly onto a C18 HPLC prep column (21 x 250 mm; Dynamax) in 100 ml of 5% acetonitrile/0.1% trifluoroacetic acid/water and eluted using a acetonitrile gradient (529%, 0.1% trifluoroacetic acid). Fractions were analyzed via electrospray mass spectrometry (API-1; Sciex) and combined and lyophilized to yield
-N-iodoacetyl-
-N-biotinyl-L-lysine trideuteromethyl ester ((M + H)+ = 555.0 (555.1 calculated)). Incorporation of the label increases the residue mass of cysteine by 429.2 Da.
d0 ICAT Reagent Synthesis
-N-iodoacetyl-
-N-biotinyl-
L-lysine methyl ester was prepared similarly using methanol (62 mg; (M + H)+ = 557.8 (558.2 calculated)). Incorporation of the label increases the residue mass of cysteine by 426.2 Da.
Labeling and Digestion of Proteins
Membrane pellets were solubilized in 0.5% SDS in 100 m
M HEPES, pH 8.5. Disulfide bonds were reduced by the addition of tributyl phosphine to a 1 mM concentration with incubation for 5 min at 90 °C. Cysteine residues were biotinylated using either polyethyleneoxide-iodoacetyl biotin (Pierce) or the ICAT reagents synthesized as above. These sulfhydryl-reactive reagents were added to a concentration of 5 mM and incubated at room temperature in the dark for 1 h. Excess reagents were removed by chloroform/methanol precipitation of the proteins (19), which were resuspended by sonication in digestion buffer (50 mM HEPES, pH 8.5), followed by stepwise addition of SDS and Triton X-100 to concentrations of 0.5 and 1%, respectively. 500 units of PNGase F (New England Biolabs) were added to deglycosylate proteins for 1 h at 37 °C. Modified trypsin (Promega) was added in a 1:50 weight ratio, and digestion was allowed to proceed overnight at 37 °C.
Avidin Purification of Biotinylated Peptides
Monomeric avidin affinity columns with a 1-ml bed volume (Pierce) were packed according to the manufacturers instructions. Protein digests were heated to 90 °C for 1 min and treated with AEBSF (Roche Molecular Biochemicals) to inhibit residual trypsin activity and loaded onto the column in phosphate-buffered saline adjusted to pH 6.5. The column was washed with 20 ml of phosphate-buffered saline to remove unlabeled peptides. An extra wash of 3 ml of deionized distilled water removed excess sodium. Biotinylated peptides were then eluted onto a poly-sulfoethyl aspartamide ion exchange column (PolyLC Inc.) with 5 ml of 50 m
M trifluoroacetic acid in 25% acetonitrile. The ion exchange column was washed with 750 µl of 0.1% formic acid/25% acetonitrile to remove residual detergent. The peptides were eluted with five 100-µl fractions of 500 mM sodium chloride/0.15% acetic acid, pH 4.1/25% acetonitrile. The fractions were then diluted to 500 µl with 0.1% heptafluorobutyric acid. The fractions were loaded onto a C18 cartridge (1 x 8 mm; Michrom BioResources) and washed with 250 µl of 0.1% heptafluorobutyric acid to remove sodium. The peptides were eluted with 20 µl of 75% acetonitrile/0.1% trifluoroacetic acid.
LC-MS/MS
Peptide mixtures (2 µl diluted to 50 µl with 0.1% heptafluorobutyric acid) were loaded onto a 0.25 x 30-mm trapping cartridge packed with Vydac 214MS low trifluoroacetic acid C4 beads. This cartridge was placed in-line with a 0.1 x 100-mm resolving column packed with Vydac 218MS low trifluoroacetic acid C18 beads. The resolving column was constructed using a PicoFritTM (New Objective) fused silica capillary pulled to a 30-µm metal-coated tip, which formed a microelectrospray ionization emitter. Peptides were eluted with a 2-h gradient of 580% (v/v) acetonitrile containing 0.1% formic acid/0.005% trifluoroacetic acid at a rate of 0.5 µl/min. Tandem mass spectrometry was performed using an ion trap instrument (LCQ DECA; ThermoFinnigan). For the mass western experiment, three or four selected precursor ions, chosen from the predicted tryptic peptides of the protein of interest, were subjected to collision-induced dissociation (CID) one after the other, cycling repeatedly throughout the LC gradient. Doubly charged ions were assumed for peptides of 8 to 20 residues. A precursor isolation window of 3 Da was used for polyethyleneoxide-iodoacetyl labeled peptides. Heavy and light ICAT-labeled peptides were simultaneously trapped and fragmented by using a 5-Da isolation window. The Sequest data base-searching program was used to generate cross-correlation scores for each CID spectrum versus the predicted peptides.
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RESULTS AND DISCUSSION
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Principles of the Technique
The mass western experimental scheme is illustrated in Fig. 1A. It is similar to the LC-MS-based approaches described above in that relatively crude cellular extracts are labeled with a cysteine-reactive biotinylation reagent and digested without prior separation followed by isolation of the cysteine-containing peptides and analysis by LC-MS/MS. But whereas the object of those experiments was to identify and quantify as many proteins as possible, a more directed approach has been taken to verify the presence of one or a few specific proteins. Given the sequence of a protein, the masses and CID fragmentation patterns of its tryptic peptides are predictable. An LC-MS/MS experiment can therefore be devised that monitors only the predicted tryptic peptides from the protein(s) of interest. The selectivity of tandem mass spectrometry allows all peptides with the wrong precursor masses to be ignored, and the specificity of the fragmentation patterns allows each chosen peptide to be distinguished from all others with the same nominal precursor mass. The number of peptides that can be analyzed in one LC-MS/MS experiment is a function of chromatographic peak widths and the duty cycle of the instrument. With chromatographic peak widths of 20 to 30 s and scan times of 5 s per CID spectrum, three or four ions can be monitored with a strong likelihood of acquiring several spectra for each of the selected ions. Wherever possible, peptides with molecular masses of 1000 to 2500 Da and lacking methionine, tryptophan, or likely sites of post-translational modification are chosen to be assayed.

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FIG. 1. The mass western procedure. A, flowchart; B, structure of the d0/d3 ICAT reagent. TBP, tributyl phosphine; PEO, polyethyleneoxide.
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The ICAT methodology was used to achieve quantitative comparisons of samples, but the reagents and scan modes were adapted to optimize sensitivity. Previously reported ICAT experiments used isotope ratios detected in full mass range scans for quantitation, with MS/MS performed in separate scans for protein identification (13, 14). A characteristic of ion trap mass spectrometers is extraordinarily high sensitivity for MS/MS experiments because of the ability to trap and accumulate precursor ions. Under the conditions used in these experiments high quality CID spectra of peptides can be obtained at the 250 attomole level, whereas the limit of detection in full mass MS mode is on the order of 5 to 10 femtomoles. Peptides can therefore be detected by MS/MS that would otherwise be lost in the background noise of full mass range MS. This property of ion traps has been exploited, for example, to obtain MS/MS data on peptides initially detected by matrix-assisted laser desorption-ionization-time-of-flight MS (20, 21). To take advantage of the sensitivity of the ion trap for MS/MS new ICAT reagents were synthesized that incorporate three (rather than eight) deuterium atoms in the heavy version (Fig. 1B) so that the multiply charged precursor ions of both heavy and light ICAT-labeled peptides are simultaneously trapped and subjected to CID. Fragment ions in the CID spectrum that contain cysteine thus appear as doublets separated by 3 Da allowing both quantitation and identification to be derived from a single scan function performed at maximal sensitivity.
Sample Handling Optimization
The intrinsic sensitivity of capillary HPLC microelectrospray ionization MS and MS/MS on ion trap mass spectrometers is extremely high; efficient sample preparation is therefore a key factor to success. The losses associated with several steps outlined in Fig. 1 were assessed. The initial protein extraction appears robust, as evidenced by minimal residue after solubilization. The extent of labeling with the ICAT reagents is difficult to determine on the membrane preparations themselves, but bovine serum albumin labeled for 30 min followed by addition of excess iodoacetamide revealed no incorporation of the second reagent (data not shown).
The removal of excess alkylating reagents after labeling is essential, because they are present in excess over proteins and otherwise dominate the mass spectra. These reagents are not removed by reverse phase, avidin, or size exclusion (nominal 10-kDa cutoff) chromatography. Reaction with tributyl phosphine yields a positively charged molecule, so cation exchange chromatography is only partially effective, although use of this method has been reported (22). Chloroform-methanol precipitation, however, effectively removed the reagents. Resolubilization of the precipitated proteins was efficient, with amino acid analysis of the labeled membrane proteins indicating 92% recovery for this procedure. Acid hydrolysis of the labeled proteins cleaves the amide bonds internal to the biotinylation reagents converting each labeled cysteine to carboxymethyl cysteine, which serves as a marker to track the abundance of labeled peptides through the analysis.
The use of SDS facilitates solubilization of the membrane proteins but poses a problem for enzymatic digestion. Trypsin activity is almost undetectable at SDS concentrations above 0.25% as measured by a synthetic substrate p-nitroaniline assay; even at 0.05% SDS, 25% of activity is lost. But if a zwitterionic or nonionic detergent like Triton X-100 is first added to an equal or greater concentration than the SDS, trypsin retains its full activity (Fig. 2A). The bands between 10 and 20 kDa observed post-digest are consistent with the presence of trypsin, and the high molecular mass smear is also found in control samples; essentially complete digestion is inferred. Purification of the labeled peptides on the avidin column is another step where sample can be lost. As determined by amino acid analysis, 82% of labeled peptides were recovered when eluted from the column. Of the peptides recovered, over 90% eluted in the second and third fractions collected (1 ml = 1 column volume per fraction), and 99% eluted in the first four fractions.

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FIG. 2. PSCA-transfected PC3 cell membrane fraction separated by SDS-PAGE. A, silver-stained gel before and after trypsin digestion. B, Western blot for PSCA; equal loading for all lanes. Lane 1, before deglycosylation; lane 2, after deglycosylation; lane 3, after precipitation and resolubilization; lane 4, following digestion with trypsin.
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Mass Western for Selective Detection of PSCA from Plasma Membranes
Prostate stem cell antigen was detected in a cell line engineered for its expression. PSCA, a 123-amino acid glycoprotein in the family of glycosylphosphatidylinositol-anchored cell surface antigens, is predominantly expressed in prostate epithelium and is overexpressed in a majority of human prostate cancers (23). PSCA is a very hydrophobic protein, having a grand average hydropathy score of 0.48, typical of membrane proteins (24). Grand average hydropathy scores in several model organisms range from -2.2 (most hydrophilic) to 1.7 (most hydrophobic) (25). Despite progress (2628), only a few proteins with positive grand average hydropathy scores have been identified by 2D electrophoresis (29, 30). PC3 cells otherwise lacking PSCA were stably transfected with the PSCA gene. An expression level of
200,000 copies per cell as estimated by Scatchard analysis (data not shown) was obtained in the cell line designated clone 11. A subcellular fraction enriched in plasma membrane was prepared from
80 million cells by differential sedimentation; one-third of the total PSCA was recovered in this fraction, with the remainder found among the cellular debris pellet as determined by Western blot (data not shown).
Proteins in the clone 11 membrane pellet were solubilized, disulfide bonds were reduced, and cysteines were labeled with commercially available polyethyleneoxide-iodoacetyl biotin. Following removal of excess reagents and lipids, proteins were deglycosylated and digested with trypsin. The course of these steps was assessed by SDS-PAGE (Fig. 2B). A Western blot probed with anti-PSCA before deglycosylation (lane 1) and after (lane 2) demonstrates consolidation of a diffuse 2530-kDa band to a compact band at 14 kDa. Precipitated protein was efficiently recovered (lane 3 versus lane 2). Native PSCA resisted trypsin digestion but was effectively cleaved following deglycosylation (lane 4). A silver-stained gel (Fig. 2A) illustrates the complexity of PC3 membrane fractions (lane 1) and completeness of digestion (lane 2).
The presence of PSCA in the clone 11 sample was proven by detection of the peptide GCSLNCVDDSQDYYVGK in the avidin-purified tryptic digest. An aliquot (10%) of the peptide mixture was analyzed by capillary reverse phase LC-microspray ion trap mass spectrometry. The instrument data system was programmed to alternately collect full mass range spectra and product ion spectra of ions with m/z 1347, the anticipated doubly charged ion of the labeled PSCA peptide. Although unnecessary for the mass western experiment, the full mass range spectra were acquired so that the overall complexity of the sample could be judged. The results are diagrammed in Fig. 3. As expected, the full mass range spectra are exceedingly complex (Fig. 3A), with many coeluting peptides at every time point. An extracted ion chromatogram for ions with m/z 1347 is also complex, with over 50 discrete peaks apparent (Fig. 3B). The PSCA peptide was distinguished from all others by its fragmentation pattern. A reconstructed ion chromatogram for ions with m/z 1347 that fragment to form a product with m/z 1189, corresponding to the y10 ion of the PSCA peptide, shows only two peaks, a small one with a retention time of 55 min and a larger peak at 64 min (Fig. 3C). The Sequest algorithm (31) was used to compare every CID spectrum to the calculated product ions of the PSCA peptide by searching a data base consisting only of the sequence of PSCA. When the cross-correlation score reported by Sequest is plotted for each CID spectrum (Fig. 3D) it is apparent that both major and minor peaks match PSCA. Examination of the complete CID spectra for both peaks revealed them to be qualitatively identical and containing extended series of ions matching those predicted for the PSCA peptide (Fig. 3E corresponds to the larger peak). The reason for the chromatographic splitting of the peptide is unknown but may be because of secondary structure effects in this relatively large and doubly-biotinylated peptide.

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FIG. 3. PSCA mass western results from transfected PC3 clone 11; detection of the tryptic peptide GCSLNCVDDSQDYYVGK. A, total ion chromatogram (TIC) for full mass range scans. B, reconstructed ion chromatogram (RIC) for all ions with m/z 1347. C, reconstructed ion chromatogram for all ions with m/z 1347 that fragment to form a product with m/z 1189 (y10 from the PSCA peptide). D, Sequest cross-correlation score versus scan number; comparison of each spectrum with the PSCA sequence. E, product ion spectrum of the peptide that eluted at 34 min. Labeled peaks indicate expected b and y series product ions.
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Validation of Quantitation from MS/MS Spectra
Reproducible quantitation of proteins by ICAT methodology was demonstrated by Gygi et al. (13) by comparison of peptide MS signals, but our approach of quantifying proteins from MS/MS data introduces new considerations. To illustrate these, lysozyme samples were labeled with the d0/d3 ICAT reagents and combined in ratios of 1:5, 1:1, and 3:1, digested, and analyzed by LC-MS/MS. The CID spectrum of the labeled tryptic peptide GYSLGNWVCAAK is displayed in Fig. 4A and is qualitatively identical for each sample. Each of the y ions beginning with y4 contains the labeled Cys, and these peaks appear as doublets from which quantitative data can be extracted. Panels B, C, and D from Fig. 4 detail the y5 ion from each sample. No chromatographic separation was observed between d0- and d3-labeled peptides, and isotope ratios were consistent across the peaks, a result consistent with all peptides studied to date. This is significant, because a shift in elution times between heavy and light isotopic forms would have a deleterious effect on quantitation. Zhang et al. (32) have measured retention time differences among d0, d3, d4, and d8 isotope labels, with the finding that d0- and d8-labeled peptides were sufficiently resolved to introduce substantial worst case errors in quantitation, with smaller deviations for d4- and d3-labeled peptides (32). Their predicted shift of about 1 s for peak maxima of d0 versus d3 would likely go undetected in our experiments, given the duty cycle of the ion trap scan functions.

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FIG. 4. Quantitation of lysozyme by ICAT LC-MS/MS analysis in three samples. A, CID spectrum of the peptide GYSLGNWVCAAK from samples containing lysozyme in a 1:5 ratio. The indicated y ions contain the ICAT label and appear as doublets in the spectrum. B, expanded view of the y5 ion from A. C, the same y5 ion as in B but from a CID spectrum of the peptide in samples containing lysozyme in a 1:1 ratio. D, likewise the y5 ion in a CID spectrum of the peptide in samples containing lysozyme in a 3:1 ratio.
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Reliable quantitation requires that the light and heavy ICAT-labeled forms of a peptide are both trapped and fragmented in the ion trap mass spectrometer. Expanding the precursor isolation window can accomplish this, at the cost of some increase in background noise. A window of 5 Da was empirically determined to be sufficient for trapping two isotope clusters separated by 1.5 Da, the difference between doubly charged ions of d0/d3-labeled peptides that contain one cysteine. If a peptide contains more than one cysteine the ion selection window is insufficiently wide. In that case either a label with fewer deuterium atoms should be used, or another peptide should be selected.
Each product ion in a CID spectrum that contains cysteine can be used to calculate relative abundance; these are averaged to obtain a more accurate measurement. In the lysozyme examples, the calculated ratios d3/d0-labeled peptides and standard deviations are 5.0 ± 0.6 for the first sample, 0.9 ± 0.1, and 0.29 ± 0.05. The relative standard deviations are 11, 9, and 18%, respectively. More precise values could be obtained by averaging the results from multiple peptides from the same protein, but even the single peptide considered here yielded precisions adequate for most biological experiments.
It is necessary that the isotope clusters of the labeled product ions are sufficiently resolved for extraction of quantitative data. The ion trap mass spectrometer operates at unit resolution, so the isotope patterns of singly charged product ions are readily apparent. A more serious complication is overlap of the second 13C isotope of a d0 ICAT-labeled product ion with the 12C isotope of the corresponding d3 ICAT-labeled product ion, skewing the apparent intensities of these peaks. The natural isotopic abundance of carbon is such that the second 13C peak is less than 10% of the 12C isotope in peptides and fragment ions below 1,000 Da, increasing to about 35% at 2,000 Da. This contribution can generally be ignored when the d3-labeled peak is more abundant than the d0-labeled peak or when the two peaks are of similar abundance. When the d3-labeled peak is small compared with the d0-labeled peak low mass product ions can be used for quantitation, because they contain little 13C, or a correction can be made based on theoretical isotope ratios. If the intention is to test hypotheses such as "protein X is more abundant in sample A than sample B," one would choose to label the putatively more abundant sample with the heavy ICAT reagent.
Quantitative Comparison of ErbB-2 Expression between Breast Tumor Cell Lines
The 185-kDa receptor tyrosine kinase ErbB2 is overexpressed in 1530% of invasive ductal breast cancers (33). A humanized monoclonal antibody raised against ErbB2 has proven an effective therapeutic for relapsed, refractory metastatic breast cancer both as a single agent and in conjunction with chemotherapy (34). Expression levels of ErbB2 have been quantified in a variety of breast tumor cell lines including MCF-7 and SK-BR-3. MCF-7 cells express
15,000 receptors per cell (clinically normal expression level) whereas SK-BR-3 cells can express as many as 2 million copies per cell, depending on culture conditions (35). Comparison of the cells used for our experiment shows an ErbB2 expression ratio of between 1:15 and 1:20 (MCF-7:SK-BR-3) by Western blot (data not shown). The quantitative version of the mass western experiment was performed to compare the relative abundance of ErbB2 in these cell lines. Membrane proteins from
50 million MCF-7 cells were labeled with the d0 ICAT reagent, and proteins from the membranes of SK-BR-3 cells were labeled with the d3 ICAT reagent. Because of the much different expression levels expected, twice as much protein as determined by bicinchonic acid assay from the MCF-7 membrane preparation was used as from the SK-BR-3 cells. The samples were mixed, precipitated, and digested with trypsin, and the cysteine-containing peptides were isolated.
Identification and relative quantification of ErbB2 was accomplished by LC-MS/MS detection of the tryptic peptide AVTSANIQEFAGCK (Fig. 5). All ions within a 5-m/z window centered on m/z 934 were subjected to CID (Fig. 5A) so that the doubly charged ions of the d0 and d3 ICAT-labeled peptide were trapped and fragmented together. A Sequest search against a data base consisting of only the ErbB2 sequence was carried out allowing for a cysteine modification of either 426.2 (the d0 ICAT reagent) or 429.2 Da (the d3 reagent). A plot of Sequest cross-correlation scores for each CID spectrum versus ErbB2 (Fig. 5B) revealed only one high scoring peptide, with a retention time of 55 min; no chromatographic separation of the heavy and light labeled forms was discernable. The CID spectra under this peak were averaged (Fig. 5C) and observed to contain the expected product ions for the ErbB2 tryptic peptide. A Sequest search of this spectrum against a data base of human proteins returned ErbB2 as the best match. Examination of the product ions that contain cysteine revealed the characteristic ICAT doublet of peaks separated by three mass units (e.g. y3 and y7 in Fig. 5, D and E). The average relative abundances of the labeled fragments (multiplied by the dilution factor) indicates an ErbB2 expression ratio of 1:16 ± 7 between MCF-7 and SK-BR-3 cells. This accords, within experimental error, with the Western blot results.

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FIG. 5. ErbB2 mass western results. A quantitative comparison of ErbB2 tryptic peptide AVTSANIQEFAGCK between SK-BR-3 and MCF-7 cells is shown. A, total ion chromatogram (TIC) for all products of m/z 934. B, Sequest cross-correlation score versus scan number; comparison of each spectrum with the ErbB2 tryptic peptide. C, product ion spectrum of the peptide eluting at 53 min. Labeled peaks indicate expected b and y series product ions. D, expanded view of y3 ion. The d0 ICAT-containing product ion at m/z 733.4 corresponds to the MCF-7-derived protein; the d3 ICAT-containing product ion at 736.4 is from the SK-BR-3-derived protein. E, expanded view of y7 ion at m/z 1208.5 and 1211.5, with assignments as in D.
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A Proteomic Approach to Hypothesis-driven Science
The mass western experiment described here represents the use of proteomic techniques for testing hypotheses about protein expression. This is in contrast to most proteomic investigations to date, which have used one- or two-dimensional electrophoresis, multidimensional protein chromatography, or ICAT LC-MS for the wholesale, non-directed collection of protein expression data (see Refs. 12, 22, and 3641 for examples). As with traditional Western blots, the mass western technique can detect a specific protein from a highly complex mixture. Rather than using an antibody for the chemical recognition of a three-dimensional epitope, physical detection of a peptide is accomplished by mass spectrometry. The specificity of the CID fragmentation pattern of a peptide is such that false-positive results are very unlikely provided the peptide chosen for detection is unique to the protein of interest, a question that can be answered by a BLAST search of the peptide sequence(s) under consideration (42). Just as blotted proteins can be probed sequentially with several antibodies, peptides from multiple proteins can be detected in subsequent LC-MS/MS experiments, provided sufficient sample is available. This experiment differs from true Western blots in that no indication of the size of a protein is obtained, because gel electrophoresis is not performed.
An important advantage of this experiment compared with techniques such as 2D electrophoresis is its general applicability. Even previously unknown proteins predicted on the basis of expressed sequence tags or genomic DNA should be identifiable if the gene sequence is correct and translated in-frame. Almost any protein that can be solubilized with SDS and reducing agents is potentially detectable. These include very large (e.g. ErbB2) and/or hydrophobic proteins such as PSCA. Some proteins (less than 10% of all proteins with molecular mass over 15 kDa) lack any cysteine residues, and some further proteins do not contain suitable Cys-containing tryptic peptides. In such cases another approach must be taken, applying alternative (e.g. amine-reactive) chemistries and fractionation principles (43). Given the sensitivity of ion trap LC-MS/MS the detection limit and dynamic range of this technique are ultimately determined by sample complexity, because incompletely resolved peptides contribute to the background noise in CID spectra. At low signal-to-noise ratios the lower abundance ICAT-labeled ion series may not be detected, placing a limit on the calculable abundance ratio. ErbB2 expressed at 15,000 copies per cell was detected, and significantly lower amounts are likely tractable with our current procedure, but many important proteins are present at copy numbers of 1,000 per cell or less. Detection of such proteins will require additional fractionation of peptides, either by selection of different peptide subsets (e.g. histidine-containing sequences) (43) or tandem chromatographic separations (8, 11, 12).
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ACKNOWLEDGMENTS
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We thank Tony Moreno and Wendy Shillinglaw in the laboratory of William J. Henzel for performing the amino acid analysis and Susan Spencer for providing the PC3 clone 11 cell line. Professor Ruedi Aebersold kindly provided advice on the avidin purification of ICAT-labeled peptides.
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FOOTNOTES
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Received, October 30, 2001, and in revised form, December 18, 2001.
Published, MCP Papers in press, December 19, 2001, DOI 10.1074/mcp.M100027-MCP200
1 The abbreviations used are: 2D, two-dimensional; AEBSF, 4-(2-aminoethyl)benzenesulfonyl fluoride; CID, collision-induced dissociation; ICAT, isotope-coded affinity tag; LC, liquid chromatography; MS, mass spectrometry; MS/MS, tandem mass spectrometry; PSCA, prostate stem cell antigen; HPLC, high pressure liquid chromatography. 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 
To whom correspondence should be addressed: Genentech, Inc., 1 DNA Way, MS #63, South San Francisco, CA 94080. Tel.: 650-225-1240; Fax: 650-225-5945; E-mail: arnott{at}gene.com.
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REFERENCES
|
---|
- Schena, M., Shalon, D., Heller, R., Chai, A., Brown, P. O., and Davis, R. W. (1996) Parallel human genome analysis: microarray-based expression monitoring of 1000 genes.
Proc. Natl. Acad. Sci. U. S. A. 93,
1061410619[Abstract/Free Full Text]
- Li, S., Rose, D. T., Kadin, M. E., Brown, P. O., and Wasik, M. A. (2001) Comparative genome-scale analysis of gene expression profiles in T cell lymphoma cells during malignant progression using a complementary DNA microarray.
Am. J. Pathol. 158,
12311237[Abstract/Free Full Text]
- Celis, J., and Gromov, P. (1999) 2D protein electrophoresis, can it be perfected?
Curr. Opin. Biotechnol. 10,
1621[CrossRef][Medline]
- Lopez, M. F. (1999) Proteome analysis I. Gene products are where the biological action is.
J. Chromatogr. B 722,
191202[CrossRef]
- Humphrey-Smith, I., Cordwell, S. J., and Blackstock, W. P. (1997) Proteome research: complementarity and limitations with respect to the RNA and DNA worlds.
Electrophoresis 18,
12171242[Medline]
- Arnott, D., Henzel, W. J., and Stults, J. T. (1998) Rapid identification of comigrating proteins by ion trap-mass spectrometry.
Electrophoresis 19,
968980[Medline]
- Davis, M. T., Beierle, J., Bures, E. T., McGinley, M. D., Mort, J., Robinson, J. H., Spahr, C. S., Yu, W., Luethy, R., and Patterson, S. D. (2001) Automated LC-LC-MS-MS platform using binary ion-exchange and gradient reversed-phase chromatography for improved proteomic analyses.
J. Chromatogr. B 752,
281291[CrossRef]
- Link, A. J., Eng, J., Scheiltz, D. M., Carmack, E., Mize, G. J., Morris, D. R., Garvick, B. M., and Yates, J. R., III (1999) Direct analysis of protein complexes using mass spectrometry.
Nat. Biotechnol. 17,
676682[CrossRef][Medline]
- Spahr, C. S., Susin, S. A., Bures, E. J., Robinson, J. H., Davis, M. T., McGinley, M. D., Kroemer, G., and Patterson, S. D. (2000) Simplification of complex peptide mixtures for proteomic analysis: reversible biotinylation of cysteinyl peptides.
Electrophoresis 21,
16351650[CrossRef][Medline]
- Davis, M. T., and Lee, T. D. (1997) Variable flow liquid chromatography-tandem mass spectrometry and the comprehensive analysis of complex protein digest mixtures.
J. Am. Soc. Mass Spectrom. 8,
10591069[CrossRef]
- Opiteck, G. J., Ramirez, S. M., Jorgenson, J. W., and Moseley, M. A., III (1998) Comprehensive two-dimensional high-performance liquid chromatography for the isolation of overexpressed proteins and proteome mapping.
Anal. Biochem. 258,
349361[CrossRef][Medline]
- Washburn, M. P., Wolters, D., and Yates, J. R., III (2001) Large-scale analysis of the yeast proteome by multidimensional protein identification technology.
Nat. Biotechnol. 19,
242247[CrossRef][Medline]
- Gygi, S. P., Rist, B., Gerber, S. A., Turecek, F., Gelb, M. H., and Aebersold, R. (1999) Quantitative analysis of complex protein mixtures using isotope-coded affinity tags.
Nat. Biotechnol. 17,
994999[CrossRef][Medline]
- Griffin, T. J., Gygi, S. P., Rist, B., Aebersold, R., Loboda, A., Jilkine, A., Ens, W., and Standing, K. G. (2001) Quantitative proteomic analysis using a MALDI quadrupole time-of-flight mass spectrometer.
Anal. Chem. 73,
978986[CrossRef][Medline]
- Anderson, L., and Seilhamer, J. (1997) A comparison of selected mRNA and protein abundances in human liver.
Electrophoresis 18,
533537[Medline]
- Gygi, S. P., Rochon, Y., Franza, B. R., and Aebersold, R. (1999) Correlation between protein and mRNA abundance in yeast.
Mol. Cell. Biol. 19,
17201730[Abstract/Free Full Text]
- Graham, J. M. (1993) in
Biomembrane Protocols I. Isolation and Analysis (Graham, J., and Higgins, J., eds) Vol. 19, pp.
97108, Humana Press, Totowa, NJ
- Hubbard, A. L., and Ma, A. (1983) Isolation of rat hepatocyte plasma membranes II, identification of membrane-associated cytoskeletal proteins.
J. Cell Biol. 96,
230239[Abstract]
- Wessel, D., and Flugge, U. I. (1984) A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids.
Anal. Biochem. 138,
141143[Medline]
- Gillece-Castro, B. L., Arnott, D., Henzel, W. J., Bourell, J. H., and Stults, J. T. (1997) Parent Ion Selection for Low Level Ion Trap MS/MS by MALDI-TOF: A Solution to the Lack of Precursor Ion Scans on the Quadrupole Ion Trap. Proceedings of the 45th ASMS Conference on Mass Spectrometry and Allied Topics, Palm Springs, California, June 15, 1997
- Zhang, X., Herring, C. J., Romano, P. R., Szczepanowska, J., Brzeska, H., Hinnebusch, A. G., and Qin, J. (1998) Identification of phosphorylation sites in proteins separated by polyacrylamide gel electrophoresis.
Anal. Chem. 70,
20502059[CrossRef][Medline]
- Ideker, T., Thorsson, V., Ranish, J. A., Christmas, R., Buhler, J., Eng, J. K., Bumgarner, R., Goodlett, D. R., Aebersold, R., and Hood, L. (2001) Integrated genomic and proteomic analyses of a systematically perturbed metabolic network.
Science 292,
929934[Abstract/Free Full Text]
- Reiter, R. E., Gu, Z., Watabe, T., Thomas, G., Szigeti, K., Davis, E., Wahl, M., Nisitani, S., Yamashiro, J., Beau, M. M. L., Loda, M., and Witte, O. (1998) Prostate stem cell antigen: a cell surface marker overexpressed in prostate cancer.
Proc. Natl. Acad. Sci. U. S. A. 95,
17351740[Abstract/Free Full Text]
- Lobry, J. R., and Gautier, C. (1994) Hydrophobicity, expressivity and aromaticity are the major trends of amino-acid usage in 999 Escherichia coli chromosome-encoded genes.
Nucleic Acids Res. 22,
31743180[Abstract]
- Wilkins, M. R., Gasteiger, E., Sanchez, J.-C., Bairoch, A., and Hochstrasser, D. F. (1998) Two-dimensional gel electrophoresis for proteome projects: the effect of protein hydropathy and copy number.
Electrophoresis 19,
15011505[Medline]
- Chevallet, M., Santoni, V., Poinas, A., Rouquie, D., Fuchs, A., Kieffer, S., Rossignol, M., Lunardi, J., Garin, J., and Rabilloud, T. (1998) New zwitterionic detergents improve the analysis of membrane proteins by two-dimensional electrophoresis.
Electrophoresis 19,
19011909[Medline]
- Pasquali, C., Fialka, I., and Huber, L. A. (1997) Preparative two-dimensional gel electrophoresis of membrane proteins.
Electrophoresis 18,
25732581[Medline]
- Molloy, M. P., Herbert, B. R., Walsh, B. J., Tyler, M. I., Traini, M., Sanchez, J.-C., Hochstrasser, D. F., Williams, K. L., and Gooley, A. A. (1998) Extraction of membrane proteins by differential solubilization for separation using two-dimensional gel electrophoresis.
Electrophoresis 19,
837844[Medline]
- Molloy, M. P., Herbert, B. R., Williams, K. L., and Gooley, A. A. (1999) Extraction of Escherichia coli proteins with organic solvents prior to two-dimensional electrophoresis.
Electrophoresis 20,
701704[CrossRef][Medline]
- Molloy, M. P. (2000) Two-dimensional electrophoresis of membrane proteins using immobilized pH gradients.
Anal. Biochem. 280,
110[CrossRef][Medline]
- Yates, J. R., III, Eng, J. K., and McCormack, A. L. (1995) Mining genomes: correlating tandem mass spectra of modified and unmodified peptides to sequences in nucleotide databases.
Anal. Chem. 67,
32023210[Medline]
- Zhang, R., Sioma, C. S., Wang, S., and Regnier, F. E. (2001) Fractionation of isotopically labeled peptides in quantitative proteomics.
Anal. Chem. 73,
51425149[CrossRef][Medline]
- Slamon, D. J., Clark, G. M., Wong, S. G., Levin, W. J., Ullrich, A., and McGuire, W. L. (1987) Human breast cancer: correlation of relapse and survival with amplification of the Her-2/neu oncogene.
Science 235,
177182[Medline]
- Cobleigh, M. A., Vogel, C. L., Tripathy, D., Robert, N. J., Scholl, S., Fehrenbacher, L., Wolter, J. W., Paton, V., Shak, S., Lieberman, G., and Slamon, D. J. (1999) Multinational study of the efficacy and safety of humanized anti-HER2 monoclonal antibody in women who have HER2-overexpressing metastatic breast cancer that has progressed after chemotherapy for metastatic disease.
J. Clin. Oncol. 17,
26392648[Abstract/Free Full Text]
- Aguilar, Z., Akita, R. W., Finn, R. S., Ramos, B. L., Pegram, M. D., Kabbinavar, F. F., Pietras, R. J., Piscane, P., Sliwkowski, M. X., and Slamon, D. J. (1999) Biologic effects of heregulin/neu differentiation factor on normal and malignant human breast and ovarian cells.
Oncogene 18,
60506062[CrossRef][Medline]
- Ji, H., Whitehead, R. H., Reid, G. E., Moritz, R. L., Ward, L. D., and Simpson, R. J. (1994) Two-dimensional electrophoretic analysis of proteins expressed by normal and cancerous human crypts: application of mass spectrometry to peptide-mass fingerprinting.
Electrophoresis 15,
391405[Medline]
- Fountoulakis, M., Juranville, J.-F., Berndt, P., Langen, H., and Suter, L. (2001) Two-dimensional database of mouse liver proteins. An update.
Electrophoresis 22,
17471763[CrossRef][Medline]
- Arnott, D., OConnell, K. L., King, K. L., and Stults, J. T. (1998) An integrated approach to proteome analysis: identification of proteins associated with cardiac hypertrophy.
Anal. Biochem. 258,
118[CrossRef][Medline]
- Simpson, R. J., Connolly, L. M., Eddes, J. S., Pereira, J. J., Moritz, R. L., and Reid, G. E. (2000) Proteomic analysis of the human colon carcinoma cell line (LIM 1215), development of a membrane protein database.
Electrophoresis 21,
17071732[CrossRef][Medline]
- Champion, K. M., Arnott, D., Henzel, W., Hermes, S., Wejkert, S., Stults, J., Vanderlaan, M., and Krummen, L. (1999) A two-dimensional protein map of Chinese hamster ovary cells.
Electrophoresis 20,
9941000[CrossRef][Medline]
- Rout, M. P., Aitchison, J. D., Suprapto, A., Hjertraas, K., Zhao, Y., and Chait, B. T. (2000) The yeast nuclear pore complex: composition, architecture, and transport mechanism.
J. Cell Biol. 148,
635651[Abstract/Free Full Text]
- Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1990) Basic local alignment search tool.
J. Mol. Biol. 215,
403410[CrossRef][Medline]
- Ji, J., Chakraborty, A., Geng, M., Zhang, X., Amini, A., Bina, M., and Regnier, F. (2000) Strategy for qualitative and quantitative analysis in proteomics based on signature peptides.
J. Chromatogr. B 745,
197210[CrossRef]