Identification of proteins in single-cell capillary electrophoresis fingerprints based on comigration with standard proteins

In the previous paper in this Journal, we reported the use of capillary sieving electrophoresis to characterize proteins expressed by single cancer cells at specific phases in the cell cycle. Analysis of the data revealed one component with cell cycle-dependent changes in expression at the 99% confidence limit. However, the amount of protein present in a single cell is far too small to allow its direct identification by mass spectrometry. In this paper, we report a method by which such proteins can be tentatively identified. We perform standard SDS-PAGE electrophoresis of the proteins contained within a homogenate prepared from an HT29 cell culture. Proteins extracted from bands in the gel are identified by mass spectrometry. The proteins also provide a set of standards that can be used to spike the sample before capillary sieving electrophoresis (CSE) separation; comigration is taken as evidence for the identity of the target protein. In a proof-of-principle experiment, a single band migrating at approximately 47 kDa was isolated from the SDS-PAGE gel generated from the HT29 cell line. Proteins extracted from this band were used to spike a CSE separation of the same extract. This band comigrated with a cell cycle-dependent component identified from single-cell analysis. In-gel digestion and LC/MS/MS were used to identify five proteins, including cytokeratin 18, which is the product of the most highly expressed gene in this cell line.     

Cell cycle-dependent protein fingerprint from a single cancer cell: image cytometry coupled with single-cell capillary sieving electrophoresis

Study of cell cycle-dependent protein expression is important in oncology, stem cell research, and developmental biology. In this paper, we report the first protein fingerprint from a single cell with known phase in the cell cycle. To determine that phase, we treated HT-29 colon cancer cells with Hoescht 33342, a vital nuclear stain. A microscope was used to measure the fluorescence intensity from one treated cell; in this form of image cytometry, the fluorescence intensity is proportional to the cell's DNA content, which varies in a predictable fashion during the cell cycle. To generate the protein fingerprint, the cell was aspirated into the separation capillary and lysed. Proteins were fluorescently labeled with 3-(2-furoylquinoline-2-carboxaldehyde, separated by capillary sieving electrophoresis, and detected by laser-induced fluorescence. This form of electrophoresis is the capillary version of SDS-PAGE. The single-cell electropherogram partially resolved approximately 25 components in a 30-min separation, and the dynamic range of the detector exceeded 5000. There was a large cell-to-cell variation in protein expression, averaging 40% relative standard deviation across the electropherogram. The dominant source of variation was the phase of the cell in the cell cycle; on average, approximately 60% of the cell-to-cell variance in protein expression was associated with the cell cycle. Cells in the G1 and G2/M phases of the cell cycle had 27 and 21% relative standard deviations in protein expression, respectively. Cells in the G2/M phase generated signals that were twice the amplitude of the signals generated by G1 phase cells, as expected for cells that are soon to divide into two daughter cells. When electropherograms were normalized to total protein content, the expression of only one component was dependent on cell cycle at the 99% confidence limit. That protein is tentatively identified as cytokeratin 18 in a companion paper.     

Instrumentation for medium-throughput two-dimensional capillary electrophoresis with laser-induced fluorescence detection

In two-dimensional capillary electrophoresis, a sample undergoes separation in the first dimension capillary by sieving electrophoresis. Fractions are periodically transferred across an interface into a second dimension capillary, where components are further resolved by micellar electrokinetic capillary electrophoresis. Previous instruments employed one pair of capillaries to analyze a single sample. We now report a multiplexed system that allows separation of five samples in parallel. Samples are injected into five first-dimension capillaries, fractions are transferred across an interface to 5 second-dimension capillaries, and analyte is detected by laser-induced fluorescence in a five-capillary sheath-flow cuvette. The instrument produces detection limits of 940 +/- 350 yoctomoles for 3-(2-furoyl)quinoline-2-carboxaldehyde labeled trypsin inhibitor in one-dimensional separation; detection limits degrade by a factor of 3.8 for two-dimensional separations. Two-dimensional capillary electrophoresis expression fingerprints were obtained from homogenates prepared from a lung cancer (A549) cell line, on the basis of capillary sieving electrophoresis (CSE) and micellar electrophoresis capillary chromatography (MECC). An average of 131 spots is resolved with signal-to-noise greater than 10. A Gaussian surface was fit to a set of 20 spots in each electropherogram. The mean spot width, expressed as standard deviation of the Gaussian function, was 2.3 +/- 0.7 transfers in the CSE dimension and 0.46 +/- 0.25 s in the MECC dimension. The standard deviation in spot position was 1.8 +/- 1.2 transfers in the CSE dimension and 0.88 +/- 0.55 s in the MECC dimension. Spot capacity was 300.     

Metabolic cytometry. Glycosphingolipid metabolism in single cells

Glycosphingolipids are found on all vertebrate cells and constitute major cell surface determinants on all nerve cells, where they contribute to cellular diversity and function. We report a method for the analysis of glycosphingolipid metabolism in single cells. The ganglioside GM1 was tagged with the fluorescent dye tetramethylrhodamine. This labeled compound was taken up and metabolized by a culture of pituitary tumor (AtT-20) cells. After 50 h, the cells were formalin fixed. Cells were aspirated into a fused-silica capillary and lysed, and components were separated by capillary electrophoresis with a laser-induced fluorescence detector. All metabolic products that retained the fluorescent dye could be detected at the low-zeptomole level. A total of 54 AtT-20 cells were individually analyzed using this procedure. The electrophoretic profiles were remarkably reproducible, which facilitated identification of components based on the migration time of fluorescently labeled standards. Eleven components were detected, and the average peak height of these components spanned more than 2 orders of magnitude, so that trace metabolites can be detected in the presence of abundant components. The most highly abundant components generated 10% relative standard deviation in normalized abundance. The average cell took up roughly 2 amol (10(6) copies) of the labeled substrate. This method allows determination of cell-to-cell diversity and regulation of glycosphingolipid metabolism.     

Desorption electrospray ionization-mass spectrometry of proteins

Desorption electrospray ionization-mass spectrometry (DESI-MS) was evaluated for the detection of proteins ranging in molecular mass from 12 to 66 kDa. Proteins were uniformly deposited on a solid surface without pretreatment and analyzed with a DESI source coupled to a quadrupole ion trap mass spectrometer. DESI-MS parameters optimized for protein detection included solvent flow rate, temperature of heated capillary tube, incident and reflection angle, sheath gas pressure, and ESI voltage. Detection limits were obtained for all protein standards, and they were found to decrease with decreasing protein molecular mass: for cytochrome c (12.3 kDa) and lysozyme (14.3 kDa) a detection limit of 4 ng/mm2 was obtained; for apomyoglobin (16.9 kDa) 20 ng/mm2; for beta-lactoglobulin B (18.2 kDa) 50 ng/mm2; and for chymotrypsinogen A (25.6 kDa) 100 ng/mm2. The DESI-MS analysis of higher molecular mass proteins such as ovalbumin (44.4 kDa) and bovine serum albumin (66.4 kDa) yielded mass spectra of low signal-to-noise ratio, making their detection and molecular weight determination difficult. In this study, DESI-MS proved to be a rapid and robust method for accurate MW determination for proteins up to 17 kDa under ambient conditions. Finally, we demonstrated the DESI-MS detection of the bacteriophage MS2 capsid protein from crude samples with minimal sample preparation.     

Gel protein capillary extraction apparatus. electronic protein transfer

A gel protein capillary extraction apparatus is developed and demonstrated for its rapid and effective transfer of SDS-protein complexes from polyacrylamide gel to a fused-silica capillary. The small dimensions of capillary columns permit the application of high voltages for achieving rapid and effective transfer of gel proteins. Furthermore, the fused-silica capillaries are internally coated with polyacrylamide for the elimination of electroosmotic pumping and protein adsorption onto the capillary wall. The extracted proteins are present in a highly concentrated solution plug as the result of field amplification and sample stacking during the extraction process. Three model proteins, including cytochrome c (14 kDa), ovalbumin (45 kDa), and beta-galactosidase (116 kDa), are visualized using coomassie blue staining and electrophoretically extracted from the gels with protein loading as low as 50 ng. The SDS-cytochrome c complexes extracted from a 50-ng protein loading are concentrated in a 30-nL solution plug inside the capillary with an estimated concentration of 0. 1 mg/mL or 10(-5) M. The capillary format allows the straightforward integration of a miniaturized trypsin-membrane reactor for on-line proteolytic digestion and ESI-MS analysis for protein/peptide identification.     

Protein quantitation using Ru-NHS ester tagging and isotope dilution high-pressure liquid chromatography-inductively coupled plasma mass spectrometry determination

An accurate, simple, and sensitive method for the direct determination of proteins by nonspecies specific isotope dilution and external calibration high-performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICPMS) is described. The labeling of myoglobin (17 kDa), transferrin (77 kDa), and thyroglobulin (670 kDa) proteins was accomplished in a single-step reaction with a commercially available bis(2,2'-bipyridine)-4'-methyl-4-carboxybipyridine-ruthenium N-succinimidyl ester-bis(hexafluorophosphate) (Ru-NHS ester). Using excess amounts of Ru-NHS ester compared to the protein concentration at optimized labeling conditions, constant ratios for Ru to proteins were obtained. Bioconjugate solutions containing both labeled and unlabeled proteins as well as excess Ru-NHS ester reagent were injected onto a size exclusion HPLC column for separation and ICPMS detection without any further treatment. A (99)Ru enriched spike was used for nonspecies specific ID calibration. The accuracy of the method was confirmed at various concentration levels. An average recovery of 100% ± 3% (1 standard deviation (SD), n = 9) was obtained with a typical precision of better than 5% RSD at 100 μg mL(-1) for nonspecies specific ID. Detection limits (3SD) of 1.6, 3.2, and 7.0 fmol estimated from three procedure blanks were obtained for myoglobin, transferrin, and thyroglobulin, respectively. These detection limits are suitable for the direct determination of intact proteins at trace levels. For simplicity, external calibration was also tested. Good linear correlation coefficients, 0.9901, 0.9921, and 0.9980 for myoglobin, transferrin, and thyroglobulin, respectively, were obtained. The measured concentrations of proteins in a solution were in good agreement with their volumetrically prepared values. To the best of our knowledge, this is the first application of nonspecies specific ID for the accurate and direct determination of proteins using a Ru-NHS ester labeling reagent.     

Sensitive and reproducible intact mass analysis of complex protein mixtures with superficially porous capillary reversed-phase liquid chromatography mass spectrometry

The compatibility of superficially porous (SP) resin for label-free intact protein analysis with online capillary LC/MS is demonstrated to give improved chromatographic resolution, sensitivity, and reproducibility. The robustness of the platform was measured against several samples of varying complexity and sample loading amount. The results indicate that capillary SP columns provide high loading capacities and that ～6 s chromatographic peak widths are typical for standard proteins in simple mixtures and proteins isolated from cell and tissue lysates. Subfemtomole detection limits for standard proteins were consistently observed, with the lowest levels at 12 amol for ubiquitin. The analysis of total heart homogenates shows that capillary SP columns provide theoretical peak capacity of 106 protein forms with 30 min total analysis time and enabled detection of proteins from complex mixtures with a single high-resolution scan. The SPLC/MS platform also detected 343 protein forms from two HeLa acid extract replicate analyses that consumed 5 × 10(4) cells and 30 min analysis time, each. Comparison of all the species observed in each HeLa replicate showed 90% overlap (309 forms) with a Pearson correlation coefficient of 89.9% for the common forms observed in the replicates. Efficient acid extract of 1 × 10(4) HeLa cells allowed reproducible detection of common modification states and members from all five of the histone families and demonstrated that capillary SPLC/MS supports reproducible label-free profiling of histones in <15 min total analysis time. The data presented demonstrate that a capillary LC/MS platform utilizing superficially porous stationary phase and a LTQ-Orbitrap FT-MS is fast, sensitive, and reproducible for intact protein profiling from small tissue and cell amounts.     

Capillary isoelectric focusing of proteins with liquid core waveguide laser-induced fluorescence whole column imaging detection

A capillary isoelectric focusing (CIEF) system with liquid core waveguide (LCW) laser-induced fluorescence whole column imaging detection was developed in this study. A Teflon AF 2400 capillary was used as both the separation channel and the axially illuminated LCW. The excitation light was introduced at one end of the capillary, and propagated forward within the capillary. As the Teflon AF 2400 capillary has a refractive index (n = 1.29-1.31) lower than that of water (n = 1.33), total internal reflection was very apparent The employment of the Teflon AF 2400 capillary avoided the use of high refractive index additives such as glycerol, accommodating the system to wider applications. Due to its inert chemical properties, the capillary exhibited limited protein adsorption and electroosmotic flow; thus, the need for capillary preconditioning with polymeric solution and the addition of polymeric additives into the sample mixture can be avoided. Three types of proteins, naturally fluorescent proteins, covalently labeled proteins, and noncovalently labeled proteins, were examined using this method. CIEF under denaturing conditions was also explored, and several advantages over the native mode were found. When compared to a commercially available instrument with UV detection, the separation efficiency and peak capacity were similar while the detection sensitivity was enhanced by 3-5 orders of magnitude.     

Biarsenical-tetracysteine motif as a fluorescent tag for detection in capillary electrophoresis

Biarsenical dyes complexed to tetracysteine motifs have proven to be highly useful fluorescent dyes in labeling specific cellular proteins for microscopic imaging. Their many advantages include membrane permeability, relatively small size, stoichiometric labeling, high affinity, and an assortment of excitation/emission wavelengths. The goal of the current study was to determine whether the biarsenical labeling scheme could be extended to fluorescent detection of analytes in capillary electrophoresis. Recombinant protein or synthesized peptides containing the optimized tetracysteine motif "-C-C-P-G-C-C-" were labeled with biarsenical dyes and then analyzed by micellar electrokinetic capillary chromatography (MEKC). The biarsenical-tetracysteine complex was stable and remained fluorescent under standard MEKC conditions for peptide and protein separations. The detection limit following electrophoresis in a capillary was less than 3 x 10(-20) mol with a simple laser-induced fluorescence system. A mixture of multiple biarsenical-labeled peptides and a protein were easily resolved. Demonstrating that the label did not interfere with bioactivity, a peptide-based enzyme substrate conjugated to the tetracysteine motif and labeled with a biarsenical dye retained its ability to be phosphorylated by the parent kinase. The feasibility of using this label for chemical cytometry experiments was shown by intracellular labeling and subsequent analysis of a recombinant protein possessing the tetracysteine motif expressed in living cells. The extension of the biarsenical-tetracysteine tag to fluorescent labeling of peptides and proteins in chemical separations is a valuable addition to biochemical and cell-based investigations.     

A paramagnetic nanoprobe to detect tumor cell death using magnetic resonance imaging

A 110 kDa (ca. 5 nm in diameter) bivalent paramagnetic nanoprobe for detecting cell death using magnetic resonance imaging (MRI) is described, in which two biotinylated C2A domains of the protein synaptotagmin-I were complexed with a single avidin molecule, which had been labeled with gadolinium chelates. This nanoprobe bound with high affinity and specificity to the phosphatidylserine exposed by dying cells and was demonstrated to allow MRI detection of apoptotic tumor cells in vitro.     

High sequence coverage of proteins isolated from liquid separations of breast cancer cells using capillary electrophoresis-time-of-flight MS and MALDI-TOF MS mapping

A method has been developed for high sequence coverage analysis of proteins isolated from breast cancer cell lines. Intact proteins are isolated using multidimensional liquid-phase separations that permit the collection of individual protein fractions. Protein digests are then analyzed by both matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) peptide mass fingerprinting and by capillary electrophoresis-electrospray ionization (CE-ESI)-TOF MS peptide mapping. These methods can be readily interfaced to the relatively clean proteins resulting from liquid-phase fractionation of cell lysates with little sample preparation. Using combined sequence information provided by both mapping methods, 100% sequence coverage is often obtained for smaller proteins, while for larger proteins up to 75 kDa, over 90% coverage can be obtained. Furthermore, an accurate intact protein MW value (within 150 ppm) can be obtained from ESI-TOF MS. The intact MW together with high coverage sequence information provides accurate identification. More notably the high sequence coverage of CE-ESI-TOF MS together with the MS/MS information provided by the ion trap/reTOF MS elucidates posttranslational modifications, sequence changes, truncations, and isoforms that may otherwise go undetected when standard MALDI-MS peptide fingerprinting is used. This capability is critical in the analysis of human cancer cells where large numbers of expressed proteins are modified, and these modifications may play an important role in the cancer process.     

Detection of green fluorescent protein in a single bacterium by capillary electrophoresis with laser-induced fluorescence

Green fluorescence protein (GFP) is a common reporter used to monitor protein expression in single cells. However, autofluorescence from endogenous components can mask the signal from GFP, particularly at low expression levels in prokaryotes. We employ capillary electrophoresis with laser-induced fluorescence for the analysis of the expression of green fluorescent protein in a single bacterium. Capillary electrophoresis separates GFP from native cellular autofluorescent components, reducing the background signal and improving detection limits. Our system provides 100 ymol (60 copies) limits of detection for GFP. To demonstrate the performance of this instrument, we employ a model system of Deinococcus radiodurans that has been engineered to express GFP under the control of the recA promoter. We report resolution and detection of GFP and autofluorescent components in a single D. radiodurans bacterium. This paper presents the first example of expression of GFP in D. radiodurans and the first detection of GFP in a single bacterium by capillary electrophoresis.     

Electrophoretic profiling of both RNA and protein from a single 250-pL sample

A novel approach is described that uses capillary electrophoresis (CE) to electrophoretically sample and separate both protein and RNA from a single injected plug of cell lysate. A 250-pL sample of lysate from Chinese hamster ovary cells (9.6 x 10(7) cells/mL) was hydrodynamically injected into a capillary containing a Tris-based aqueous buffer. This was followed by selective electrokinetic ejection of RNA from the lysate into water, yielding an effective cell concentration of RNA of 3000 cells/mL. The cellular components (e.g., proteins) retained in the capillary were separated and then detected with laser-induced fluorescence (LIF) using 275-nm excitation. The ejected/diluted sample was subsequently injected into a separate CE-LIF system, which utilized an entangled polymer sieving matrix and 543-nm excitation for the detection of ethidium bromide-labeled nucleic acids (i.e., RNA). Virtually no sample preparation is required other than simple washing and lysing of the cells isolated from culture. This combined approach can be easily modified for the detection of any analyte through adjustment of CE-HF conditions. In addition, it provides an effective method for desalting cellular RNA samples having complex matrixes, which results in improved RNA injection efficiency and a 7600-fold effective signal enhancement over total lysate injection.     

Processing complex mixtures of intact proteins for direct analysis by mass spectrometry

For analysis of intact proteins by mass spectrometry (MS), a new twist to a two-dimensional approach to proteome fractionation employs an acid-labile detergent instead of sodium dodecyl sulfate during continuous-elution gel electrophoresis. Use of this acid-labile surfactant (ALS) facilitates subsequent reversed-phase liquid chromatography (RPLC) for a net two-dimensional fractionation illustrated by transforming thousands of intact proteins from Saccharomyces cerevisiae to mixtures of 5-20 components (all within approximately 5 kDa of one another) for presentation via electrospray ionization (ESI) to a Fourier transform MS (FTMS). Between 3 and 13 proteins have been detected directly using ESI-FTMS (or MALDI-TOF), and the fractionation showed a peak capacity of approximately 400 between 0 and 70 kDa. A probability-based identification was made automatically from raw MS/MS data (obtained using a quadrupole-FTMS hybrid instrument) for one protein that differed from that predicted in a yeast database of approximately 19,000 protein forms. This ALS-PAGE/RPLC approach to proteome processing ameliorates the "front end" problem that accompanies direct analysis of whole proteins and assists the future realization of protein identification with 100% sequence coverage in a high-throughput format.     

Probing proteomes using capillary isoelectric focusing-electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry.

Unlike the genome, the proteome is exquisitely sensitive to cellular conditions and will consist of proteins having abundances dependent upon stage in the cell cycle, cell differentiation, response to environmental conditions (nutrients, temperature, stress etc.), or disease state(s). Therefore, the study of proteomes under well-defined conditions can provide a better understanding of complex biological processes and inference of protein function. Thus, much faster, more sensitive, and precise capabilities for the characterization of cellular constituents are desired. We describe progress in the development and initial application of the powerful combination of capillary isoelectric focusing (CIEF) and Fourier transform ion cyclotron resonance (FTICR) mass spectrometry for measurements of the proteome of the model system Escherichia coli. Isotope depletion of the growth media has been used to improve mass measurement accuracy, and the comparison of CIEF-FTICR results for the analysis of cell lysates harvested from E. coli cultured in normal and isotopically depleted media are presented. The initial studies have revealed 400-1000 putative proteins in the mass range 2-100 kDa from total injections of approximately 300 ng of E. coli proteins in a single CIEF-FTICR analysis.     

Proteome Analysis of Thyroid Cancer Cells After Long-Term Exposure to a Random Positioning Machine

Annulling gravity during cell culturing triggers various types of cells to change their protein expression in a time dependent manner. We therefore decided to determine gravity sensitive proteins and their period of sensitivity to the effects of gravity. In this study, thyroid cancer cells of the ML-1 cell line were cultured under normal gravity (1?g) or in a random positioning machine (RPM), which simulated near weightlessness for 7 and 11?days. Cells were then sonicated and proteins released into the supernatant were separated from those that remained attached to the cell fragments. Subsequently, both types of proteins were fractionated by free-flow isoelectric focussing (FF-IEF). The fractions obtained were further separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) to which comparable FF-IEF fractions derived from cells cultured either under 1?g or on the RPM had been applied side by side. The separation resulted in pairs of lanes, on which a number of identical bands were observed. Selected gel pieces were excised and their proteins determined by mass spectrometry. Equal proteins from cells cultured under normal gravity and the RPM, respectively, were detected in comparable gel pieces. However, many of these proteins had received different Mascot scores. Quantifying heat shock cognate 71?kDa protein, glutathione S-transferase P, nucleoside diphosphate kinase A and annexin-2 by Western blotting using whole cell lysates indicated usefulness of Mascot scores for selecting the most efficient antibodies.     

Use of capillary electrophoresis and endogenous fluorescent substrate to monitor intracellular activation of protein kinase A

Here we demonstrate for the first time the use of an endogenous multiphosphorylatable substrate for monitoring the intracellular activation of kinase with capillary electrophoresis. First, we devised a novel PCR-based strategy for controlled generation of short multirepeat DNA sequences and applied this method to generate a green fluorescence protein (GFP)-tagged protein substrate containing eight phosphorylation sites for protein kinase A (PKA). The protein substrate was transiently expressed in C2C12 rat myoblast cells, and intracellular PKA was then activated by adding [8]-bromo-cyclic AMP to the cell culture medium. Phosphorylated product and nonphosphorylated substrate present in the crude cell extract were separated by capillary zone electrophoresis and detected with laser-induced fluorescence of the GFP tag. The identities of two electrophoretic peaks were confirmed by both phosphorylation of the substrate and dephosphorylation of the product in vitro. The proposed method was applied to monitoring the activation of PKA in single myoblast cells. It advantageously allowed us to avoid microinjection of the substrate, the procedure that is both hard to perform and excessively invasive when applied to small mammalian cells.     

Identification of components of protein complexes using a fluorescent photo-cross-linker and mass spectrometry

This study describes a novel method for improving the specific recognition, detection, and identification of proteins involved in multiprotein complexes. The method is based on a combination of coimmunoprecipitation, chemical cross-linking, and specific fluorescent tagging of protein components in close association with one another. Specific fluorescent tagging of the protein complex components was achieved using the cleavable, fluorescent cross-linker sulfosuccinimidyl 2-(7-azido-4-methylcoumarin-3-acetamido) ethyl-1,3'-dithiopropionate (SAED). Following dissociation and separation by SDS-PAGE, the fluorescently tagged proteins are then visualized by UV illumination, excised, and, following in-gel digestion, identified by mass spectrometry. In this study, a complex of the HIV-envelope protein gp120 and its cellular receptor CD4 was used as a model system. The sensitivity of detection of fluorescent SAED-labeled proteins in SDS gels, and the sensitivity of the mass spectrometric identification of fluorescent proteins after in-gel digestion, is in the range of a few hundred femtomoles of protein. This sensitivity is comparable to that achieved with silver-staining techniques, but fluorescence detection is protein independent and no background interference occurs. Furthermore, fluorescence labeling is significantly more compatible with mass spectrometric identification of proteins than is silver staining. The first application of this strategy was in the investigation of the mechanism of spermiation, the process by which mature spermatids separate from Sertoli cells. For the coimmunoprecipitation experiment, an antibody against paxillin, a protein involved in spermatid-Sertoli cell junctional complexes, was used. More components of the paxillin protein complex were visible by fluorescence detection of SAED-labeled proteins than were visible on comparable silver-stained gels. Mass spectrometric analysis of the fluorescently labeled proteins identified integrin alpha6 precursor as a protein associated in a complex with paxillin. The identification of integrin alpha6 precursor was confirmed by Western blot analysis and verifies the applicability of this novel approach for identifying proteins involved in protein complexes.