Identification of rat liver glutathione S-transferase Yb subunits by partial N-terminal sequencing after electroblotting of proteins onto a polyvinylidene difluoride membrane from an analytical isoelectric focusing gel

Rat liver glutathione S-transferases were partially purified using S-hexyl glutathione affinity chromatography, followed by native isoelectric focusing employing a pH 7-11 or pH 3-10 gradient. Proteins were excised and eluted from the gel for determination of subunit composition using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In separate experiments, isoelectric focusing gels were equilibrated with a sodium dodecyl sulfate-containing buffer at high pH, and proteins on the gel were electroblotted onto a polyvinylidene difluoride membrane, utilizing graphite plates as electrodes. The membrane-bound proteins were visualized by Coomassie Brilliant Blue staining. The protein bands were then excised from the membrane and inserted into a gas phase sequenator for direct sequencing. N-Terminal sequences thus determined were compared with published cDNA sequences. The isoelectric points (pIs) and positions on the isoelectric focusing gel of Yb1Yb1, Yb1Yb2 and Yb2Yb2 subunits were determined. We have also located on the pH 3-10 focusing gel an N-terminal blocked glutathione S-transferase which has a molecular weight similar to Yb subunits.     

Computerized, comprehensive databases of cellular and secreted proteins from normal human embryonic lung MRC-5 fibroblasts: identification of transformation and/or proliferation sensitive proteins

Databases of protein information from human embryonal lung fibroblasts (MRC-5) have been established using computer analyzed two-dimensional gel electrophoresis. One thousand four hundred and eighty-two cellular proteins (1060 with isoelectric focusing and 422 with nonequilibrium pH gradient electrophoresis, in the first dimension) ranging in molecular mass between 8 and 234 kDa were separated and numbered. Information entered in the database (in most cases for major proteins) includes: protein name, HeLa protein catalog number, mouse protein catalog number, proteins matched in transformed human epithelial amnion cells (AMA) and peripheral blood mononuclear cells (PBMC), transformation and/or proliferation sensitive proteins, synthesis in quiescent cells, cell cycle regulated proteins, mitochondrial and heat shock proteins, cytoskeletal proteins and proteins whose synthesis is affected by interferons. Additional information entered for a few transformation-sensitive proteins that have been selected for future studies includes levels of synthesis and amounts in fetal human tissues. A total of four hundred and seventy-six [35S]methionine labeled polypeptides (258 isoelectric focusing; 218, nonequilibrium pH gradient electrophoresis) secreted by MRC-5 fibroblasts were separated and recorded (J. E. Celis et al., Leukemia 1987, 1, 707-717). Information entered in this database includes molecular weight and transformation sensitive proteins. These databases, as well as those of epithelial and lymphoid cell proteins (J. E. Celis et al., Leukemia 1988, 9, 561-601), represent the initial stages of a systematic effort to establish comprehensive databases of human protein information. In the long run, these databases are expected to offer a useful framework in which to focus the human genome sequencing effort.     

Immunochemical-based method for detection of hazelnut proteins in processed foods

A competitive enzyme-linked immunosorbent assay (ELISA) was developed to detect hazelnut by using polyclonal antibodies generated against a protein extract of roasted hazelnut. No cross-reactivity was observed in tests against 39 commodities, including many common allergens, tree nuts, and legumes. Hazelnut protein standard solutions at 0.45 ng/mL [inhibition concentration (IC80) of the competitive test] were clearly identified by the ELISA. An extraction and quantification method was developed and optimized for chocolate, cookies, breakfast cereals, and ice cream, major food commodities likely to be cross-contaminated with undeclared hazelnut during food processing. No sample cleanup was required when extracts were diluted 10-fold. Recovery results were generated with blank matrixes spiked at 4 levels from 1 to 10 microg/g hazelnut protein. With the developed extraction and sample handling procedure, hazelnut proteins were recovered at 64-83% from chocolate and at 78-97% from other matrixes. A confirmatory technique was developed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western transfer. The developed methods were applied to a small market survey of chocolate products and allowed the identification of undeclared hazelnut in these products.     

Identification of Tuber borchii Vittad. mycelium proteins separated by two-dimensional polyacrylamide gel electrophoresis using amino acid analysis and sequence tagging

This paper reports the first results in the proteome analysis of Tuber borchii Vittad. mycelium, an ectomycorrhizal fungus poorly defined genetically, but known for its generation of edible fruit bodies known as white truffles. Employing isoelectric focusing on immobilized pH gradients, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, we obtained an electropherogram presenting over 800 spots within the window of isoelectric points (pI) 3.5-9 and a molecular mass of 10-200 kDa. Different reducing agents were tested in the sample preparation buffers, and the standard lysis buffer plus 2% w/v polyvinylpolypyrrolidone allowed the best solubilization and resolution of the proteins. The T. borchii proteins separated in micropreparative gels were electroblotted onto polyvinylidene difluoride membranes and visualized by Coomassie staining. Twenty-three proteins were excised and analyzed by the combination of amino acid and N-terminal analysis. One protein was identified by matching its amino acid composition, estimated isoelectric point and molecular mass against the SWISS-PROT and EMBL databases. Four spots were successfully tagged by Edman microsequencing but no homologous sequences were found in databases.     

Protein mapping of peanut extract with capillary electrophoresis

Protein separation can be achieved with different modes of capillary electrophoresis, such as with capillary gel electroporesis (CGE) or with capillary zone electrophoresis (CZE). CZE protein mapping of peanut extract was approached in four different ways, combining neutral-coated or multilayer-coated capillaries with pHs well over or under the isoelectric point range of the proteins of interest. At acidic pHs, the mobility ranges of the major peanut allergens Ara h1, Ara h2, Ara h3, and Ara h6 were identified. Although the pH is a major factor in CZE separation, buffers with different compositions but with the same pH and ionic strength showed significantly different resolutions. Different components of the electrolyte were studied in a multifactorial design of experiment. CE-SDS and CZE proved to be suitable for protein mapping and we were able to distinguish different batches of peanut extract and burned peanut extract.     

Preparative isoelectric focusing of reduced wheat gluten proteins

Proteins extracted from gluten of the bread wheat cultivar Fiorello 2 in the presence of 2-mercaptoethanol or dithiothreitol were separated by isoelectric focusing in a free solution in a pH 3-10 gradient containing 50% v/v 1-propanol or urea. The collected fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in 10% gels (high and medium molecular weight glutenin subunits) and 16% gels (low molecular weight gliadins). The isoelectric focusing pattern of gluten polypeptides in 50% v/v 1-propanol was comparable to that obtained on two-dimensional gel electrophoresis, based on isoelectric focusing and polyacrylamide gel electrophoresis or nonequilibrium pH gradient electrophoresis and polyacrylamide gel electrophoresis. A similar isoelectric focusing pattern was also observed when 3M urea was used as solvent. New gluten polypeptides, similar in mobility to the high molecular weight subunits of glutenin were detected at acidic pH.     

Several small GTP-binding proteins are strongly down-regulated in simian virus 40 (SV40) transformed human keratinocytes and may be required for the maintenance of the normal phenotype

High resolution two-dimensional (2-D) gel electrophoresis in combination with the blot overlay nucleotide binding assay was used to reveal low molecular weight GTP-binding proteins expressed by primary cultured, normal human keratinocytes. Forty one small GTP-binding proteins (30 isoelectric focusing, IEF; and 11 nonequilibrium pH gradient electrophoresis, NEPHGE) ranging in molecular weights from 18000 to 30000 and isoelectric points from 4.4 to 8.0 were detected and mapped in the master human keratinocyte database. Four GTP-binding proteins were identified by 2-D gel immunoblotting and these correspond to rap 1 and 2 and two forms of rab6. ras Proteins are most likely present in the [α³²P]GTP 2-D gel blots but their levels may be too low to be detected by immunoblotting. Quantitative changes in the relative expression levels of [α³²P]GTP-binding proteins in normal proliferating and simian virus 40 (SV40) transformed human keratinocytes (K 14) were determined by scintillation counting of the radioactive spots excised from the nitrocellulose blots. The results showed that thirteen of these proteins were not expressed in transformed K14 keratinocytes, implying that they may play a role in the maintenance of the normal cell phenotype.     

Effect of heat stress on tomato fruit protein expression

We delayed the ripening of tomato fruit for several days (average 5 days) by a 1-day heat treatment at 37-42 degrees C. We analyzed the tomato fruit pericarp proteins, which were altered by the heat stress, using two-dimensional electrophoresis. Heat stress caused about 23.7% of the proteins in the pericarp to disappear and about 1.1% of new proteins to appear. We determined their apparent molecular mass, isoelectric point, and N-terminal amino acid sequence. Identified proteins included antioxidant enzymes, heat shock proteins, cell-wall-related proteins, etc.     

Comparison of ethanol-soluble proteins from different rye (Secale cereale) varieties by two-dimensional electrophoresis

The major storage proteins from six rye varieties, grown under the same conditions in 1997 and 1998 in Rønhave, Denmark, were analyzed by two‐dimensional (2‐D) polyacrylamide gel electrophoresis. The proteins were extracted from ground rye kernels with 70% ethanol and separated by 2‐D electrophoresis. The gels were scanned, compared using ImageMaster® software and the data sets were analyzed by principal component analysis (PCA) using THE UNSCRAMBLER software. Afterwards MATLAB was used to make a cluster analysis of the varieties based on PCA. The analysis of the gels showed, that the protein patterns (number of different proteins and their isoelectric points and molecular weights) from the six rye varieties were different. Based on the presence of unique cultivar‐specific spots it was possible to differentiate between all six varieties if the two harvest years were investigated separately. When the results were combined from the two years five varieties could be differentiated. The results from the PCA confirmed the finding of the unique spots and cluster analysis was made in order to illustrate the results. The combination of the results from 2‐D electrophoresis and other grain characteristics showed that one protein spot was located close to the parameters bread volume and bread height.     

Differences between predicted and observed sequences in Saccharomyces cerevisiae

We recently studied the protein composition of a Saccharomyces cerevisiae wine yeast strain (K310) of enological interest. About 2,500 spots of 8-250 kDa observed molecular mass were resolved by two-dimensional gel electrophoresis. Experimental molecular masses and isoelectric points were calculated for most of them. Twenty-seven proteins were subjected to Edman microsequencing. N-terminal sequences of 12/27 proteins were determined, whereas internal sequences of 6/27 proteins were obtained following in situ proteolysis. Comparison between the experimental data and those reported in the SWISS-PROT database revealed some differences between genotypic and phenotypic sequences. These are indicative of the changes a protein can undergo with respect to the primary structure coded by the genomic DNA. Our results highlight the need to complement genomic analysis with detailed proteomics in order to refine the vast amount of information provided by DNA sequencing and to find an exact correlation between genome and proteome.     

Effect of Sample Preparation on the Detection and Quantification of Selected Nuts Allergenic Proteins by LC-MS/MS

The detection and quantification of nut allergens remains a major challenge. The liquid chroma-tography tandem mass spectrometry (LC-MS/MS) is emerging as one of the most widely used methods, but sample preparation prior to the analysis is still a key issue. The objective of this work was to establish optimized protocols for extraction, tryptic digestion and LC-MS analysis of almond, cashew, hazelnut, peanut, pistachio and walnut samples. Ammonium bicar-bonate/urea extraction (Ambi/urea), SDS buffer extraction (SDS), polyvinylpolypyrroli-done (PVPP) extraction, trichloroacetic acid/acetone extraction (TCA/acetone) and chloro-form/methanol/sodium chloride precipitation (CM/NaCl) as well as the performances of con-ventional tryptic digestion and microwave-assisted breakdown were investigated. Overall, the protein extraction yields ranged from 14.9 ± 0.5 (almond extract from CM/NaCl) to 76.5 ± 1.3% (hazelnut extract from Ambi/urea). Electrophoretic profiling showed that the SDS extraction method clearly presented a high amount of extracted proteins in the range of 0–15 kDa, 15–35 kDa, 35–70 kDa and 70–250 kDa compared to the other methods. The linearity of the LC-MS methods in the range of 0 to 0.4 µg equivalent defatted nut flour was assessed and recovery of internal standards GWGG and DPLNV(d8)LKPR ranged from 80 to 120%. The identified bi-omarkers peptides were used to relatively quantifier selected allergenic protein form the inves-tigated nut samples. Considering the overall results, it can be concluded that SDS buffer allows a better protein extraction from almond, peanut and walnut samples while PVPP buffer is more appropriate for cashew, pistachio and hazelnut samples. It was also found that conventional overnight digestion is indicated for cashew, pistachio and hazelnut samples, while microwave assisted tryptic digestion is recommended for almond, hazelnut and peanut extracts.     

Interferon gamma regulates a unique set of proteins in fresh human bladder transitional cell carcinomas

Poly(A) mRNA was isolated from human placental trophoblast cells stimulated with 100 U/mL of interleukin-2 and 5 microg/mL of phytohemagglutinin and reverse-transcribed. The cDNA coding for the mature interferon-gamma (IFN-gamma) protein was amplified using specific primers, cloned into the pGEX-4T2 vector, and expressed in Escherichia coli. Treatment of four fresh bladder transitional cell carcinoma (TCC) biopsies (TCCs 845-1, grade II, Ta; TCC 925-1, grade II, Ta; TCC 919-1, grade III, T1; TCC 950-1, grade III, T1) with the purified recombinant trophoblast IFN-gamma (50 U/mL, 20 h), followed by proteome analysis using two-dimensional gel electrophoresis, revealed several major proteins whose level of expression were affected by this cytokine. Of these, five (tryptophanyl-tRNA synthetase, the interferon gamma-inducible protein gamma3, mangase superoxide dismutase, and two unknown proteins of apparent molecular masses of 35.8 and 11.2 kDa, respectively) were upregulated in at least 75% of the tumors analyzed while one was downregulated (aldose reductase). Proteins were identified using a combination of techniques that included microsequencing, two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) immunoblotting and comparison with the transitional cell carcinoma isoelectric focusing (IEF) database (http://biobase.dk/cgi-bin/celis). Proteome profile analysis of primary cultures from a low-grade lesion (TCC 846-1, Grade II, Ta) labeled in the presence and absence of IFN-gamma showed that all of the proteins disregulated in vivo were also affected in the cultures. The cultured cells, on the other hand, exhibited additional changes that were not detected in vivo and that may reflect adaptation to the culturing conditions. Taken together, the results provide a first glance at the effect of IFN-gamma on the protein expression profiles of TCCs, and in due course may form the basis for more comprehensive studies aimed at evaluating the usefulness of this cytokine in bladder cancer management.     

Analysis of intact proteins from cerebrospinal fluid by matrix-assisted laser desorption/ionization mass spectrometry after two-dimensional liquid-phase electrophoresis

A novel combination of methods, two-dimensional liquid-phase electrophoresis (2D-LPE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS), have been used for the analysis of intact brain-specific proteins in cerebrospinal fluid (CSF). 2D-LPE is especially useful for isolating proteins present in low concentrations in complex biological samples. The proteins are separated in the first dimension by liquid-phase isoelectric focusing (IEF) in the Rotofor cell and in the second dimension by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) in the Preparative cell. The removal of SDS by chloroform/methanol/water, followed by sample preparation with the addition of n-octylglucoside, easily interfaced 2D-LPE with MALDI-TOFMS for analysis of intact proteins. Further characterization by proteolytic digestion is also demonstrated. The knowledge of both the molecular weights of the protein and of the proteolytic fragments obtained by peptide mapping increases specificity for protein identification by searching in protein sequence databases. Two brain-specific proteins in human CSF, cystatin C and transthyretin, were isolated in sufficient quantity for determination of the mass of the whole proteins and their tryptic digest by MALDI-TOFMS. This approach simplified the interface between electrophoresis and MALDI-TOFMS.     

Commercial lateral flow devices for rapid detection of peanut (<Emphasis Type="Italic">Arachis hypogaea)</Emphasis> and hazelnut (<Emphasis Type="Italic">Corylus avellana)</Emphasis> cross-contamination in the industrial production of cookies

Lateral flow devices (LFDs) are qualitative immunochromatographic tests for the rapid and specific detection of target analytes. We investigated commercially available LFDs for their ability to detect potentially allergenic peanut and hazelnut in raw cookie dough and chocolate, two important food matrices in the industrial production of cookies. Each three commercial LFDs for the detection of hazelnut and peanut were performed according to the manufacturers’ instructions. All LFDs had comparably satisfactory specificity that was investigated with a variety of characteristic foods and food ingredients used in the production of cookies. In concordance with hazelnut-specific enzyme-linked immunosorbent assays (ELISAs), walnut was the most cross-reactive food for hazelnut-specific LFD. The sensitivity was verified in raw cookie doughs and chocolates that were either spiked with peanut or hazelnut between 1 and 25 mg/kg, respectively. Two hazelnut-specific LFDs detected hazelnut at a level of 3.5 mg/kg in both matrices, whereas the third LFD detected hazelnut at a level of 3.9 mg/kg in dough and 12.5 mg/kg in chocolate. Two peanut-specific LFDs detected peanut at a level of 1 mg/kg in both matrices. The third LFD detected peanut at a level of 14.2 mg/kg in chocolate and 4 mg/kg in dough. In conclusion, specific and sensitive LFD were identified for each hazelnut and peanut, having a level of sensitivity that is comparable to commercial ELISA for the investigated matrices. Such sensitive, specific, and rapid tests are useful analytical tools for allergen screening and sanitation in the industrial manufacture of foods.     

Single-cell protein analysis of a single mouse embryo by two-dimensional capillary electrophoresis

The characterization of protein expression from a single-cell mouse embryo using two-dimensional capillary electrophoresis (2D-CE) is described. These zygotes were obtained from Hsf1 gene knockout mice. Single zygotes were lysed off-column and proteins were fluorescently labeled using the fluorogenic dye 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ). After injection, analytes were separated first according to molecular weight using capillary sieving electrophoresis (CSE) and then by micellar electrokinetic capillary chromatography (MEKC) to obtain protein expression fingerprints. Analytes were detected in a sheath flow cuvette using laser-induced fluorescence. In a 1-h 2D-CE separation, over 100 components were resolved with a spot capacity of 380.     

An immunoblotting method for high-resolution isoelectric focusing of protein isoforms on immobilized pH gradients

Post-translational modifications such as phosphorylation and acetylation are important elements for regulating the activity of enzymes or structural proteins. These modifications give rise to isoforms that are often not resolved by separation methods relying on the size of proteins. Here, we optimized an isoelectric focusing (IEF)-immunoblotting method suitable for analyzing protein isoforms in total cell extracts. The separations were carried out in parallel on commercially available immobilized pH gradient slab gels (IPG). The buffer used for separation contained urea, thiourea, dithiothreitol, as well as the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate (CHAPS), and was designed to match those used in two-dimensional polyacrylamide gel electrophoresis (PAGE) separations where efficient solubilization is required. Proteins were transferred to membranes by passive diffusion in the presence of 4 M guanidinium chloride using protocols optimized for several protein classes (tubulin, stathmin, 14-3-3 proteins) some of which required removal of CHAPS prior to transfer. In conjunction with narrow-range pH gradient gels, excellent resolution of isoforms differing by phosphorylation or acetylation was achieved. The usefulness of pI and titration curve calculations for predicting the pI shifts expected for post-translational modifications of proteins with known amino acid composition was demonstrated. Using stathmin--which contains four phosphorylation sites--as an example, the effects on the pI-shifts were well predicted. This sensitive and widely applicable IEF-blotting technology is expected to be especially suited for analyzing protein isoforms first detected by two-dimensional electrophoresis.     

Esophageal carcinoma-associated proteins detected by two-dimensional polyacrylamide gel electrophoresis

Patterns of proteins of five surgically resected esophageal carcinomas were studied by two-dimensional polyacrylamide gel electrophoresis with silver staining. The samples of normal esophageal mucosa and esophageal carcinoma from the same patient were compared. Each gel had ca. 300 protein spots and had a similar pattern of proteins. Four spots were observed in all of the esophageal carcinomas that were not present in any of the normal mucosae. The molecular weights and isoelectric points were 46,000 and 5.3, 46,000 and 5.2, 36,000 and 4.7 and 33,000 and 5.1, respectively. One spot was observed in all of the normal mucosae but not in any of the esophageal carcinomas. Its molecular weight and isoelectric point were 27,000 and 5.3, respectively.     

Studies on the selenoproteome in the cultured cells of lung and trachea by gel electrophoretic techniques

In the present studies radiotracer techniques have been combined with biochemical separation procedures to investigate the selenium-containing proteins in the culture cells of the lung, trachea and their subcellular fractions. Subcellular separation of the lung and trachea tissues has been achieved by differential ultracentrifugation. The selenium-containing proteins in these compartments have been investigated by labeling of lung and trachea cultured cells in vitro with Se-75, gel electrophoretic separation of the proteins and autoradiographic detection of the tracer. The protein separation by gel electrophoresis using mono-dimensional (1D)- and two-dimensional (2D)-SDS-PAGE has been successfully applied for the selenium research. It has resulted in the detection of a large number of selenium-containing proteins. Two-dimensional gel electrophoresis (2-DE) was also helpful in the identification of the proteins of interest according to their molecular mass and isoelectric point. In this way more than 30 selenium-containing proteins could be distinguished in the lung and trachea samples. Some of them such as Gpx1, Trx1, SelP, SelT and Sel15 could be identified by means of immunoassays, their molecular weight and pI values and localized in the cellular compartments.     

Preparative separation of immunoglobulins from bovine colostrum by continuous divergent-flow electrophoresis

Immunoglobulins in bovine colostrum were separated and fractionated from other proteins using the method and instrumentation developed in our laboratory. The proposed separation was based on bidirectional isotachophoresis/moving boundary electrophoresis with electrofocusing of the analytes in a pH gradient from 3.9 to 10.1. The preparative instrumentation included the trapezoidal non-woven fabric that served as separation space with divergent continuous flow. The defatted and casein precipitate-free colostrum supernatant was loaded directly into the instrument without any additional colostrum pre-preparation. Immunoglobulin G was fractionated from other immune proteins such as bovine serum albumin, β-lactoglobulin, and α-lactalbumin, and was continuously collected in separated fractions over 3 h. The fractions were further processed, and isolated immunoglobulin G in the liquid fractions was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by re-focusing in gel isoelectric focusing. Separated immunoglobulin G was detected in seven fractions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a gradually decreased concentration in the fractions. Re-focusing of the proteins in the fractions by gel isoelectric focusing revealed multiple separated zones of immunoglobulin G with the isoelectric point values covering the range from 5.4 to 7.2. Each fraction contained distinct zones with gradually increased isoelectric point values and decreased concentrations from fraction to fraction.     

Analysis of glycosyl phosphatidylinositol-anchored proteins by two-dimensional gel electrophoresis

The aim of this study was to characterize mammalian glycosyl phosphatidylinositol (GPI)-anchored proteins y two-dimensional gel electrophoresis using immobilized pH gradients. Analysis was performed on detergent-resistant membrane fractions of baby hamster kidney (BHK) cells, since such fractions have previously been shown to be highly enriched in GPI-anchored proteins. Although the GPI-anchored proteins were readily separated by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), these proteins were undetectable on two-dimensional (2-D) gels, even though these gels unambiguously revealed high enrichment of known hydrophobic proteins of detergent-resistant membranes such as caveolin-1 and flotillin-1 (identified by Western blotting and tandem mass spectrometry, respectively). Proper separation of GPI-anchored proteins required cleavage of the lipid tail with phosphatidylinositol-specific phospholipase C, presumably to avoid interference of the hydrophobic phospholipid moiety of GPI-anchors during isoelectric focusing. Using this strategy, BHK cells were observed to contain at least six GPI-anchored proteins. Each protein was also present as multiple isoforms with different isoelectric points and apparent molecular weights, consistent with extensive but differential N-glycosylation. Pretreatment with N-glycosidase F indeed caused the different isoforms of each protein to collapse into a single spot. In addition, quantitative removal of N-linked sugars greatly facilitated the detection of heavily glycosylated proteins and enabled sequencing by nanoelectrospray-tandem mass spectrometry as illustrated for the GPI-anchored protein, Thy-1.