Improved Protein Identification through the Use of Unstained Gels David R. Loiselle,†,| William R. Thelin,‡,| Carol E. Parker,†,| Nedyalka N. Dicheva,† Barry A. Kesner,‡ Viorel Mocanu,† Frank Wang,§ Sharon L. Milgram,‡ Maria R. Esteban Warren,† and Christoph H. Borchers*,† Department of Biochemistry & Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, Department of Cell & Developmental Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, and BioMachines, Inc., Carrboro, North Carolina Received November 23, 2004
Abstract: In this work, a method for improved protein identification of low-abundance proteins using unstained gels, in combination with robotics and matrix-assisted laser desorption/ionization tandem mass spectrometry, has been developed and evaluated. Omitting the silverstaining process resulted in increased protein identification scores, an increase in the number of peptides observed in the MALDI mass spectrum, and improved quality of the tandem mass spectrometry data. Keywords: unstained gels • protein identification • PAGE • silver staining • immunoprecipitation • robotics • automation • MALDIMS
Introduction In conventional proteomics, mass spectrometry is used to identify proteins separated on polyacrylamide gels, which are frequently stained with Coomassie, fluorescent dyes, or silver stain. The choice of protein stains can greatly influence the success of subsequent protein identification by mass spectrometry. Coomassie is very compatible with MALDI-MS and ESI-MS, while fluorescent dyes such as Sypro Ruby (Molecular Probes Inc., Eugene, OR)1,2 are more sensitive than Coomassie, and are fairly compatible with MS,3 but are less sensitive than silver staining,4 which has problems of protein-to-protein variability,5 and limited dynamic range.6 In addition, silver ions appears to reduce the mass spectrometric sensitivity of MALDIMS and ESI-MS.7,8 In an effort to circumvent these problems, modified silverstaining protocols have been developed.1,7,8 These additional steps can also reduce the amount of protein remaining in the gel,9 and the chemicals commonly used for destaining silverstained gels and are often incompatible with mass spectrometry and must be removed prior to analysis, while hydrogen * To whom correspondence should be addressed. Department of Biochemistry & Biophysics, University of North Carolina at Chapel Hill, 402 Mary Ellen Jones Building CB #7260, Chapel Hill, NC 27599Phone: (919) 843-5310, Fax: (919) 966-2852. E-mail:
[email protected]. † Department of Biochemistry & Biophysics, University of North Carolina at Chapel Hill. ‡ Department of Cell & Developmental Biology, University of North Carolina at Chapel Hill. § BioMachines, Inc.. | Authors contributed equally to this work.
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peroxide can lead to methionine oxidation. Even with these modified techniques, silver staining development times are often shortened to reduce problems associated with overstaining.4 This, in itself, can lead to low-abundance species being undetected. Some researchers have developed techniques using the native fluorescence of the proteins themselves,10 which would eliminate the problems of staining and destaining entirely. Recently, Roegener, et al have coupled this technique to ingel digestion and mass spectrometric identification.11 However, the gel bands must still be visualized by UV detection, and the technique is limited by the background fluorescence of the gels. In this paper, the MS results are compared for proteins extracted from silver-stained and unstained 1D-PAGE gels, using a robotic gel cutter to excise multiple gel plugs from selected regions of the gel. Significant improvements in protein detection and identification were achieved on a mixture of standard proteins using this technique, which does not rely on visualization of the protein bands. This method, of course, generates a large number of gel plugs which may or may not contain protein, and therefore requires robotics and highthroughput mass spectrometry. This approach, however, is particularly useful for analyzing the protein complement from co-immunoprecipitation or pull-down experiments where there is a wide dynamic range in protein abundance. We have demonstrated the advantages of using unstained gels in the analyses of two “real-world” samples. In these examples, proteins were confidently identified only when cut and digested from unstained gels.
Experimental Section Protein Standards. Commercial molecular weight markers for 1D gels were used as the protein standards for these experiments. The mixture contains the following proteins: myosin (188 kDa), β-galactosidase (116 kDa), phosphorylase-b (98 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (28 kDa), trypsin inhibitor (21.5 kDa), lysozyme (14 kDa), and aprotinin (6 kDa). Variable concentrations of the standards were prepared by diluting a stock solution of Biorad broad range SDS mass markers (2 µg each protein/µL) with Biorad SDS Reducing Sample Buffer (Biorad, Hercules, CA). The working standard solutions were incubated for 10 min at 100 °C then loaded in amounts of 2000, 200, 20, and 2 ng (of each protein in the marker mixture) onto 10.1021/pr049785o CCC: $30.25
2005 American Chemical Society
technical notes alternating wells of two duplicate 1D gels. Because of potential difficulties in locating the unstained gel bands, prestained markers (Amersham High-Range Rainbow Marker prestained Molecular Weight markers, GE Healthcare, Piscataway, NJ) were run in the outermost and center lanes. 1-D SDS-PAGE of Protein Standards. 1D SDS-PAGE was performed simultaneously on two duplicate gels following the Laemmli12 procedure and using pre-cast 10% Tris-Glycine mini gels and Tris/glycine/SDS running buffer (Invitrogen Corporation, Carlsbad, CA). Following electrophoresis, the gels were immersed for 30 min in an aqueous fixative solution containing 10% ethanol and 7% acetic acid. One of the gels was silverstained with reagents from Invitrogen’s Silver Quest kit using a modified staining protocol.13 The second gel was stored in the fixative solution until further processing. Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Immunoprecipitation. Membranes prepared from forty 100 mm dishes of airway epithelial cells (CalU3) were solubilized in binding buffer containing 50 mM Tris pH 7.6, 150 mM NaCl, 0.2% CHAPS, and 1X Roche complete protease inhibitor cocktail (Roche Diagnostics Corporation, Indianapolis, IN). CFTR was immunoprecipitated from the solubilized membranes using a CFTR affinity matrix (anti-CFTR monoclonal antibody 570 covalently cross-linked to protein G Dynabeads (Dynal Biotech Inc., Brown Deer, WI)). Bound proteins were eluted using CFTR-disaggregation buffer14 and resolved on a 4-20% acrylamide SDS-PAGE gel. Proteins were either visualized by silver staining (Invitrogen), or the gel was fixed but not stained. The region of the gel corresponding to the molecular weight of CFTR was excised and digested in-gel with trypsin. YAP65 Co-immunoprecipitation. Mouse M1 cells were transfected using lipofectamine plus (Invitrogen) with a plasmid coding for N-terminally HA-tagged human YAP-65 protein. Cells were lysed in RIPA buffer (25 mM Tris, pH 7.4, 150 mM KCl, 5 mM EDTA, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS), which was diluted 1:5 with binding buffer (50 mM Tris (pH 7.6), 150 mM NaCl, 10 mM EDTA, 0.1% Triton X-100, and protease inhibitor cocktail (Roche). HA-YAP65 was immunoprecipitated using a HA-affinity matrix (Roche), bound proteins were eluted using 1X disaggregation buffer, and resolved on a 7.5% acrylamide SDS-PAGE gel. Bound proteins were either visualized by silver staining (Invitrogen) as described above, or the gel was fixed but not stained. Gel Excision and Digestion. Gel plugs were excised using a robotic system (BioMachines 2DiD benchtop Gel Processing System from Leap Technologies, Carrboro, NC). This system uses software capable of cutting gel plugs from an entire lane using a uniform programmed pattern. This uniformity reduces variability and maximizes the “cut” area of the gel to include as much sample as possible. For the gel containing standard proteins, the robot was programmed to use a cutting pattern in which a row of four 1.3 mm diameter plugs alternated with a row of three plugs. Cuts from the alternating rows were staggered rather than stacked to include as much gel material as possible. Gel plugs cut from each row were combined into a single well for further processing. The prestained markers served as a visual guide of the boundaries in which protein may be present. For the lanes containing protein standards an area of gel within these boundaries was selected for excision, although the entire lane from top to bottom could have been cut if desired. Ideally, proteins would separate in a lane that is nearly straight from
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top to bottom. In reality, the lane may shift slightly to the left or right, which would be problematic in cutting unstained gels. The prestained markers that were run on the flanking and center lanes serve as good indicators for these shifts. The cutting pattern can then be adjusted accordingly. The cut plugs were further processed using a Genomic Solutions ProGest robotic workstation (Genomic Solutions, Ann Arbor, MI), using a previously described method.13,15 Briefly, after a series of washes, the excised gel bands were digested with trypsin at 37 °C for 8 h, after which the tryptic peptides were extracted. The peptide extract was lyophilized overnight and resuspended in 5 µL of 50:50 methanol:0.1% aqueous formic acid immediately prior to spotting on the MALDI target. Mass Spectrometric Analysis and Database Searching. MALDI-MS/MS data was acquired using an ABI Voyager 4700 MALDI-TOF/TOF mass spectrometer (Applied Biosystems, Inc., Framingham, MA). For the ABI TOF/TOF analyses, a saturated solution of recrystallized R-cyano 4-hydroxycinnamic acid (Aldrich, St. Louis, MO) in 50:50 acetonitrile: 40 mM ammonium citrate/ 0.1% trifluoroacetic acid was used as the matrix solution. A 0.3 µL aliquot of each sample was spotted followed by 0.3 µL of matrix solution, and allowed to dry at room temperature. MS and MS/MS spectra were acquired in automated mode in which the 8 most intense peaks with a signal-to noise ratio greater than 25 were selected by the 4700 Data Explorer software for MS/MS analysis. The peptide mass fingerprinting and sequence tag data from the TOF/TOF was evaluated using ABI’s GPS Explorer scores, which are derived from Mascot searches.16 Ion Scores were generated by submitting MS/MS spectra to the NCBI database via the Mascot search engine. Only proteins with Ion Scores greater than 20 were included in our analysis and evaluation since Ion Scores below 20 are not considered statistically significant by Mascot.
Results and Discussion Experiments with Protein Standards. To examine the effect of silver staining on protein identification at different levels of protein, pairs of gels were prepared at four different levels of standard proteins. Silver staining was performed on one of these two gels (Figure 1A), while the other gel was left unstained. In the unstained gel, prestained markers flanking the sample lanes were used to determine the position of the sample-containing lanes. During staining, the silver stain development was monitored and was stopped when the most abundant proteins (proteins in the 2000 ng lane) were visible. This was done both to prevent overstaining, and to produce a gel that simulates a “real world” sample, which often contains a mixture of proteins with a wide range of protein levels. This under-development produces a silver-stained gel in which the highly abundant proteins are clearly visible while lower abundant proteins are not visible. As shown in Figure 1A, proteins are clearly visible at the 2000 and 200 ng levels, slightly visible at 20 ng and invisible at the 2 ng level. In Figure 1B this same gel is shown after excision of the gel plugs. For each lane the alternating “4+3” cut pattern followed by the BioMachines 2DiD robot can clearly be seen. Figure 1C shows an image of the unstained gel, where only the prestained markers are visible. Only nine protein bands were expected from this mixture, but a visual examination of the stained gel (Figure 1A, Lane 1) reveals more bands than expected, probably Journal of Proteome Research • Vol. 4, No. 3, 2005 993
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Improved Protein Identification Using Unstained Gels
Table 1. Comparison of the Three ABI GPS Explorer “Quality” Factors Stained and Unstained Gels by Summing the Scores for All Proteins in Each Lanea peptide count protein score total ion score (summed by lane) (summed by lane) (summed by lane)
2000 ng Ratio (Unstained/ Stained) 200 ng Ratio (Unstained/ Stained) 20 ng Ratio (Unstained/ Stained)
1.08
1.28
1.44
1.41
1.46
1.43
2.77
2.91
2.59
a The quality parameters for the protein “hits” in each lane are summed, and the ratios of these scores for the unstained versus the stained gel are calculated. Higher scores mean a higher degree of confidence in the identification.
Table 2. Unstained/Stained Ratios for the Three ABI “Quality” Parameters, summed by proteina
Figure 1. 1-D PAGE analysis of protein standards, with 2000, 200, 20, and 2 ng of each protein loaded in Lanes 1-4, respectively. Serial dilutions of protein molecular weight markers were loaded onto different lanes of the gel. (A) Silver-stained gel. (B) Silverstained gel following excision by the BioMachines 2DiD gel excision robot, showing where the gel plugs had been removed. The gel image illustrates the 4 × 3 cut pattern of gel plugs generated by the 2DiD robot. (C) Unstained gel with stained molecular weight markers for reference. Stained markers were used on the unstained gel in the flanking and center lanes to help visualize the position of the lanes and assist in the automated cutting of the bands.
due to the high amount of protein loaded and degradation of some of the proteins. For each row of plugs, the database search results from the ABI GPS Explorer software was examined for significant “hits” for the known protein identities. Three “data quality” parameters resulting from the ABI Data Explorer search: Peptide Count, Protein Score, and Total Ion Score were evaluated only for these significant hits. Peptide Count is the total number of (nonredundant) peptides detected for that protein (i.e., the number of peptides that match a theoretical digest of the protein identified.) The Protein Score is based on MS and MS/ MS data, and represents a combination of the peptide mass fingerprinting (MS data) results and the MS/MS data (Ion Scores). Mascot Ion Scores reflect the quality of the MS/MS spectra, and the Total Ion Score is based on weighted Ion Scores (MS/MS data) for the individual peptides. Data for the 2000, 200, and 20 ng levels are shown, however no significant hits were found for Lane 4, the 2 ng level, so these data are not included. 994
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protein (2000 ng)
peptide count
protein score
total ion score
Myosin β-galactosidase Phosphorylase-b Serum Albumin Ovalbumin Carbonic Anhydrase Lysozyme Aprotinin Trypsin Inhibitor
1.12 0.92 0.90 1.21 1.65 0.94 1.68 ∞a 0.66
1.69 1.35 0.98 2.10 3.02 0.83 1.61 ∞ 0.67
2.99 1.69 1.01 2.76 3.70 1.01 1.50 ∞ 0.68
protein (200 ng)
peptide count
protein score
total ion score
Myosin β-galactosidase Phosphorylase-b Serum Albumin Ovalbumin Carbonic Anhydrase Lysozyme Trypsin Inhibitor
1.49 0.82 1.82 1.56 1.04 1.14 4.50 0.77
2.33 0.63 1.75 1.97 1.26 1.36 6.52 0.66
2.47 0.86 1.55 2.06 1.18 1.77 6.75 0.59
protein (20 ng)
peptide count
protein score
total ion score
Myosin Serum Albumin Ovalbumin Lysozyme Carbonic Anhydrase Trypsin Inhibitor
∞ ∞ 1.00 2.50 2.00 1.38
∞ ∞ 1.43 5.94 3.31 1.02
∞ ∞ 1.38 11.65 9.09 0.81
a Unstained/Stained Ratios for the Three ABI “Quality” Parameters, summed by protein*. *∞ ) divide by zero; protein not detected in stained gel.
For a quantitative evaluation, these Mascot results are presented in three different ways. Although a true quantitative comparison is difficult, because these quality scores are not truly “additive”, some indication of the relative improvement resulting from the use of unstained gels can be seen in Tables 1 and 2. Table 1 shows the ratios of the sums of the scores of all of the standard proteins within each band. The ratios show that the scores are higher for all three parameters (Peptide Count, Protein Score, and Total Ion Score) for samples from unstained gels than from silver-stained gels. All levels showed improve-
technical notes ment due to the absence of silver stain, and the improvement was greatest (by almost a factor of 3) at the lowest concentration. Another way of examining the data is to sum all of the scores by protein, no matter where it is found on the gel. This would compensate for a band which was accidentally “split” during the cutting of the unstained gel, and also would compensate for protein degradation which often occurs in biological samples. As stated above, even the proteins in our standard mixture appear to have undergone partial degradation before they were spotted on the gel. This resulted in identification of the same protein in multiple bands within the same lane. Myosin, for example was found in gel bands ranging from the expected molecular weight of 188 kDa, down to 14 kDa. Comparisons of the total scores for each protein are presented in Table 2. As expected, the individual quality scores for both stained and unstained gels decrease with lower sample loadings. Although, there is clearly variation between proteins, the highest ratios (i.e., the most improvement in scores) are observed at the lowest sample loading. Overall, 19 of 27 quality parameters (70%) were higher in the unstained than the stained gel at 2000 ng sample loading. Similarly, 18 of 24 quality parameters (75%) were higher in the unstained gel than the stained gel at 200 ng, and 16 out of 18 quality parameters (89%) were higher in the unstained gel than the stained gel at 20 ng. Overall, this study demonstrates the use of unstained gels is most beneficial at the lower sample levels, where the number of peptides from the stained gels was often insufficient for peptide mass fingerprinting and the peptide abundances were too low for sequencing. These are, of course, the levels where improvements in protein identification are most needed. Even though “blind” cutting may result in an unstained band being split between two wells, it is more than offset by the increase in scores resulting from the absence of silver stain. Two components in the protein mixture were protease inhibitors (aprotinin, and trypsin inhibitor), and these behaved differently from the other proteins studied. Aprotinin, the smallest protein, was only observed at the highest loading (2000 ng), and then only in the unstained gel. This may be due to the difficulty in recovering peptides from small proteins. At the higher sample loadings, trypsin inhibitor was the only protein that had significantly lower quality scores from the unstained gel as compared with the silver-stained gel. Since trypsin was used to lyse the proteins before extraction from the gel, a possible explanation is that within the gel the inhibitor retains some activity and that the silver-staining process may impair its ability to function as a trypsin inhibitor. This could lead to less inhibition of trypsin by the silver-stained inhibitor than from the unstained inhibitor. Application to Biological Problems. The experiments with the protein standards demonstrated the utility of this approach for identification of low-abundance proteinssproteins that are at levels approaching the sensitivity of silver staining. Although there are many potential applications where this method would be useful, co-immunoprecipitation experiments present an ideal biological sample to test the validity of this approach. In each of the two examples below, the ultimate goal of each experiment was to immunoprecipitate a target protein (either CFTR or YAP65) from cell lysates and identify other proteins that are directly associated. These samples were chosen to test this method as they had proven challenging to analyze by MS due to their inherent low signal-to-noise ratios. Contaminants such as nonspecifically associated proteins and antibodies
Loiselle et al.
Figure 2. Comparison of peptide mass fingerprinting results obtained from the in-gel digestion of stained and unstained gels containing immunoprecipitated proteins of airway epithelial cells. The areas within the boxes on the stained gel (A) and the unstained gel (B) as indicated by the arrows were excised, digested with trypsin and analyzed by MALDI-MS. Red asterisks in the MALDI-MS spectra indicate peptides matching CFTR.
contribute to high experimental background. Additionally, the co-immunoprecipitated proteins are often purified at low abundances as they are expressed at low levels or interact with low affinity in vivo. CFTR Sample. In this example, our technique allowed the successful identification of CFTR from an immunoprecipitation sample that could not be unambiguously identified from the corresponding silver-stained gel. CFTR, the protein mutated in cystic fibrosis (CF), functions as an epithelial chloride channel, and mutations in CFTR cause defects in channel biosynthesis, trafficking, and regulationsprocesses that may be mediated by protein-protein interactions. Thus, associated proteins may provide drug targets to treat CF. Endogenous CFTR is expressed at low levels in epithelial tissues, thus requiring large amounts of epithelial cells to purify a sufficient amount of channel for subsequent proteomic analyses. To test the feasibility of this approach, CFTR was immunoprecipitated from airway epithelial cells. CFTR immune-complexes were separated by SDS-PAGE gel electrophoresis, and were either stained with silver stain (Invitrogen), or fixed and left unstained. Figure 2 shows a comparison of peptide mass fingerprinting data from the silver-stained and unstained gels (the red asterisks indicate peptides matching CFTR). Cutting and digesting the unstained gel resulted in a mass spectrum in which more CFTR peptides were detected compared to the silverstained sample (9 and 5 CFTR peptides, respectively) and with greater signal intensity. This increased signal intensity and more complete sequence coverage (4% for the silver-stained samples versus 8% for the unstained sample) resulted in a confident identification of CFTR. YAP65-Associated Protein. YAP65 (Yes-kinase Associated Protein, mw ∼65 kDa) is a scaffolding protein that compartmentalizes signaling molecules in the cytoplasm and functions as a transcriptional co-activator in the nucleus.17 Since YAP functions as a scaffold, identifying the proteins it associates with is fundamental to understanding the cellular role of YAP. To identify these proteins, HA-tagged YAP was immunoprecipitated from mouse epithelial cell lysates. The bound proteins were separated and visualized with either Coomassie or silver stain. Journal of Proteome Research • Vol. 4, No. 3, 2005 995
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Figure 3. Identification of a YAP-associated protein from an unstained gel. The YAP-associated protein labeled as p100 was not visible in the silver-stained SDS-PAGE gel (A). A large area of an unstained gel was cut into plugs, which were combined into “bands”, digested, and analyzed by MALDI-MS (B). Performing this procedure on the unstained gel allowed the identification of a YAP-associated protein, p100. Red asterisks indicate peptides matching p100. Peaks labeled with “T” are trypsin autolysis peaks.
While the abundant protein YAP65 could easily be detected by Coomassie staining, no coprecipitating proteins could be visualized, even by highly sensitive, non-MS compatible silver staining. Despite this, the YAP65 immunoprecipitate was separated by PAGE, and the entire unstained gel lane was cut, digested, and analyzed by MALDI-TOF/TOF (Figure 3). This approach allowed us to identify a putative YAP65 interacting protein, designated p100. This protein was not visible by silver staining (Figure 3A), and was only detected by blindly cutting a large portion of the gel band into gel plugs, digesting these plugs, and analyzing the digests on a MALDITOF/TOF mass spectrometer. The mass spectrum obtained from gel plugs containing this protein was of sufficient quality to obtain a match using the Mascot database-searching program. Peptides shown with a red asterisk (Figure 3B) correspond to predicted peptides from p100. Relying on visual or fluorescent detection of additional proteins in this sample would have resulting in nondetection of p100. This again demonstrates the utility of this method for the analysis of biologically relevant samples.
Conclusions Overall, improvements in all three “quality” parameters from the ABI GPS Explorer database searching program (Peptide Count, Protein Score, and Total Ion Score) were observed for proteins extracted from unstained vs stained gels. These higher scores correspond to improved peptide mass fingerprinting and peptide sequencing data, and this improvement was most notable at low levels, where improvement is most critical for 996
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successful protein identification. Although this “increase” in sensitivity is, in reality, a reduction in the loss of sensitivity due to silver staining, it can make the difference between successful identification of an in-gel digested protein versus no identification. The increase in peptide abundance resulted in automated selection of additional peptides for MS/MS acquisition, thereby reducing the need for manual acquisition of MS/MS data, which is often required for silver-stained gels. This approach is particularly useful for analyzing the protein complement from co-immunoprecipitation or pull-down experiments where a wide dynamic range of “bait” protein and interacting proteins are present. The ultimate application of this technique would be to cut and digest an entire lane of a 1-D gel, or even an entire unstained 2-D gel. As demonstrated by the detection of p100 in this paper, this technique will result in the identification of many more proteins than would be detected from a comparable silver-stained gel. This approach will lead to a large number of samples/gel plugs to analyze, but with the availability of high throughput techniques such as robotic gel cutting, automated in-gel digestion, MALDI-MS/MS, and automated database searching, this type of experiment is clearly feasible. We are already using this technique to detect low levels of protein in a limited region of the gelsfor example, a region of the gel where a Western blot analysis has indicated a target protein may be present. Abbreviations. HA, haemagglutinin; RIPA, radioimmunoprecipitation.
Acknowledgment. This work was supported by a gift from an anonymous donor for research targeted to Proteomics and Cystic Fibrosis, and by a grant from the Cystic Fibrosis Foundation (CFFTI STUTTS01U0 for CB, SM), NIH (5R21ES011997-02 for CB), NIH (P30ES10126 for CB, MW), NIH (5P30CA016086-30 for DL, CB, ND, CP, MW), NIH (2P01HL045100-11 for V.M.). We also thank Dr. Jack Riordan for the gift of the antiCFTR antibody. References (1) Lopez, M. F.; Berggren, K.; Chernokalskaya, E.; Lazarev, A.; Robinson, M.; Patton, W. F. A comparison of silver stain and Sypro ruby protein gel stain with respect to protein detection in two-dimensional gels and identification by peptide mass profiling. Electrophoresis 2000, 21, 3673-3683. (2) Malone, J. P.; Radabaugh, M. R.; Leimgruber, R. M.; Gerstenecker, G. S. Practical aspects of fluorescent staining for proteomic applications. Electrophoresis 2001, 22, 919-932. (3) Lanne, B.; Panfilov, O. Protein staining influences the quality of mass spectra obtained by peptide mass fingerprinting after separation on 2-D gels. A comparison of staining with coomassie brilliant blue and Sypro ruby. J. Proteome Res. 2005, 4, 175179. (4) Patton, W. F. Detection technologies in proteome analysis. J. Chromatogr. B 2002, 771, 3-31. (5) Merrill, C.; Goldman, D.; Van Keuren, M. Methods Enzymol. 1984, 104, 441-447. (6) Syrovy, I.; Hodny, Z. Staining and quantification of proteins separated by polyacrylamide gel electrophoresis. J. Chromatogr. 1991, 569, 175-196. (7) Scheler, C.; Lamer, S.; Pan, Z.; Li, X. P.; Salnikow, J.; Jungblut, P. Peptide mass fingerprint sequence coverage from differently stained proteins on two-dimensional electrophoresis patterns by matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS). Electrophoresis 1998, 19, 918-927. (8) Shevchenko, A.; Wilm, M.; Vorm, O.; Mann, M. Mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels. Anal. Chem. 1996, 68, 850-858. (9) Harry, J. L.; Wilkins, M. R.; Herbert, B. R.; Packer, N. H.; Gooley, A. A.; Williams, K. L. Proteomics: Capacity versus utility. Electrophoresis 2000, 21, 1071-1081.
technical notes (10) Sluszny, C.; Yeung, E. S. One- and two-dimensional miniaturized electrophoresis of proteins with native fluorescence detection. Anal. Chem. 2004, 76, 1359-1365. (11) Roegener, J.; Lutter, P.; Reinhardt, R.; Blueggel, M.; Meyer, H. E.; Anselmetti, D. Ultrasensitive detection of unstained proteins in acrylamide gels by 2003native UV fluorescence. Anal. Chem. 2003, 75, 157-159. (12) Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage t4. Nature 1970, 227, 680685. (13) Parker, C. E.; Warren, M. R.; Loiselle, D. R.; Dicheva, N. N.; Borchers, C. H. “Identification of components of protein complexes” In Methods in Molecular Biology; Patterson, W. C., Ed.; Humana Press: Patterson, NJ, 2004; Vol. in press. (14) Sarkadi, B.; Price, E. M.; Boucher, R. C.; Germann, U. A.; Scarborough, G. A. Expression of the human multidrug re-
Loiselle et al. sistance cDNA in insect cells generates a high activity drugstimulated membrane atpase. J. Biol. Chem. 1992, 267, 48544858. (15) Borchers, C.; Peter, J. F.; Hall, M. C.; Kunkel, T. A.; Tomer, K. B. Identification of in-gel digested proteins by complementary peptide-mass fingerprinting and tandem mass spectrometry data obtained on an electrospray ionization quadrupole time-of-flight mass spectrometer. Anal. Chem. 2000, 72, 1163-1168. (16) www.matrixscience.com. (17) Mohler, P. J.; Kreda, S. M.; Boucher, R. C.; Sudol, M.; Stutts, M. J.; Milgram, S. L. Yes-associated protein 65 localizes p62(c-yes) to the apical compartment of airway epithelia by association with ebp50. J. Cell Biol. 1999, 147, 879-890.
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