Strategy for identifying protein-protein interactions of gel-separated proteins and complexes by mass spectrometry

A strategy for identifying and characterizing protein interactions among gel-separated proteins and complexes has been developed and tested. The method involves the efficient recovery of proteins or complexes from native gels without affecting their conformational integrity. The use of limited proteolysis of protein complexes, isolated from the gel or formed from the interaction of gel-recovered proteins with potential binding partners, has enabled local binding domains to be efficiently identified using a combination of microfiltration and mass spectrometric analysis. The application of mass spectrometry affords high detection sensitivities, enabling the strategy to be applied to low levels of protein and protein mixtures. The approach is demonstrated for both antigen-antibody and peptide-protein complexes for which protein-binding regions are characterized among simple peptide mixtures and proteolytic digests. The strategy can be easily adapted to achieve high sample throughput and automation using gel-excision robotics and provides a means to study protein interactions in complex biological mixtures and extracts.     

Specificity of fatty acid acylation of cellular proteins

Labeling of the BC3H1 muscle cell line with [3H] palmitate and [3H]myristate results in the incorporation of these fatty acids into a broad spectrum of different proteins. The patterns of proteins which are labeled with palmitate and myristate are distinct, indicating a high degree of specificity of fatty acylation with respect to acyl chain length. The protein-linked [3H]palmitate is released by treatment with neutral hydroxylamine or by alkaline methanolysis consistent with a thioester linkage or a very reactive ester linkage. In contrast, only a small fraction of the [3H]myristate which is attached to proteins is released by treatment with hydroxylamine or alkaline methanolysis, suggesting that myristate is linked to proteins primarily through amide bonds. The specificity of fatty acid acylation has also been examined in 3T3 mouse fibroblasts and in PC12 cells, a rat pheochromacytoma cell line. In both cells, palmitate is primarily linked to proteins by a hydroxylamine-labile linkage while the major fraction of the myristic acid (60-70%) is linked to protein via amide linkage and the remainder via an ester linkage. Major differences were noted in the rate of fatty acid metabolism in these cells; in particular in 3T3 cells only 33% of the radioactivity incorporated from myristic acid into proteins is in the form of fatty acids. The remainder is presumably the result of conversion of label to amino acids. In BC3H1 cells, palmitate- and myristate-containing proteins also exhibit differences in subcellular localization. [3H]Palmitate-labeled proteins are found almost exclusively in membranes, whereas [3H]myristate-labeled proteins are distributed in both the soluble and membrane fractions. These results demonstrate that fatty acid acylation is a covalent modification common to a wide range of cellular proteins and is not restricted solely to membrane-associated proteins. The major acylated proteins in the various cell lines examined appear to be different, suggesting that the acylated proteins are concerned with specialized cell functions. The linkages through which fatty acids are attached to proteins also appear to be highly specific with respect to the fatty acid chain length.     

Proteomic analysis of synaptosomes using isotope-coded affinity tags and mass spectrometry

Synaptosomes are isolated synapses produced by subcellular fractionation of brain tissue. They contain the complete presynaptic terminal, including mitochondria and synaptic vesicles, and portions of the postsynaptic side, including the postsynaptic membrane and the postsynaptic density (PSyD). A proteomic characterisation of synaptosomes isolated from mouse brain was performed employing the isotope-coded affinity tag (ICAT) method and tandem mass spectrometry (MS/MS). After isotopic labelling and tryptic digestion, peptides were fractionated by cation exchange chromatography and cysteine-containing peptides were isolated by affinity chromatography. The peptides were identified by microcapillary liquid chromatography-electrospray ionisation MS/MS (muLC-ESI MS/MS). In two experiments, peptides representing a total of 1131 database entries were identified. They are involved in different presynaptic and postsynaptic functions, including synaptic vesicle exocytosis for neurotransmitter release, vesicle endocytosis for synaptic vesicle recycling, as well as postsynaptic receptors and proteins constituting the PSyD. Moreover, a large number of soluble and membrane-bound molecules serving functions in synaptic signal transduction and metabolism were detected. The results provide an inventory of the synaptic proteome and confirm the suitability of the ICAT method for the assessment of synaptic structure, function and plasticity.     

Quantitative profiling of differentiation-induced microsomal proteins using isotope-coded affinity tags and mass spectrometry

An approach to the systematic identification and quantification of the proteins contained in the microsomal fraction of cells is described. It consists of three steps: (1) preparation of microsomal fractions from cells or tissues representing different states; (2) covalent tagging of the proteins with isotope-coded affinity tag (ICAT) reagents followed by proteolysis of the combined labeled protein samples; and (3) isolation, identification, and quantification of the tagged peptides by multidimensional chromatography, automated tandem mass spectrometry, and computational analysis of the obtained data. The method was used to identify and determine the ratios of abundance of each of 491 proteins contained in the microsomal fractions of na?ve and in vitro- differentiated human myeloid leukemia (HL-60) cells. The method and the new software tools to support it are well suited to the large-scale, quantitative analysis of membrane proteins and other classes of proteins that have been refractory to standard proteomics technology.     

Protein cross-linking analysis using mass spectrometry, isotope-coded cross-linkers, and integrated computational data processing

Distance constraints in proteins and protein complexes provide invaluable information for calculation of 3D structures, identification of protein binding partners and localization of protein-protein contact sites. We have developed an integrative approach to identify and characterize such sites through the analysis of proteolytic products derived from proteins chemically cross-linked by isotopically coded cross-linkers using LC-MALDI tandem mass spectrometry and computer software. This method is specifically tailored toward the rapid analysis of low microgram amounts of proteins or multimeric protein complexes cross-linked with nonlabeled and deuterium-labeled bis-NHS ester cross-linking reagents (both commercially available and readily synthesized). Through labeling with [18O]water solvent and LC-MALDI analysis, the method further allows the possible distinction between Type 0 and Type 1 or Type 2 modified peptides (monolinks and looplinks or cross-links), although such a distinction is more readily made from analysis of tandem mass spectrometry data. When applied to the bacterial Colicin E7 DNAse/Im7 heterodimeric protein complex, 23 cross-links were identified including six intersubunit cross-links, all between residues that are close in space when examined in the context of the X-ray structure of the heterodimer. In addition, cross-links were successfully identified in five single subunit proteins, beta-lactoglobulin, cytochrome c, lysozyme, myoglobin, and ribonuclease A, establishing the generality of the approach.     

A dataset of human liver proteins identified by protein profiling via isotope-coded affinity tag (ICAT) and tandem mass spectrometry

Proteins from human liver carcinoma Huh7 cells, representing transformed liver cells, and cultured primary human fetal hepatocytes (HFH) and human HH4 hepatocytes, representing nontransformed liver cells, were extracted and processed for proteome analysis. Proteins from stimulated cells (interferon-alpha treatment for the Huh7 and HFH cells and induction of hepatitis C virus [HCV] proteins for the HH4 cells) and corresponding control cells were labeled with light and heavy cleavable ICAT reagents, respectively. The labeled samples were combined, trypsinized, and subject to cation-exchange and avidin-affinity chromatographies. The resulting cysteine-containing peptides were analyzed by microcapillary LC-MS/MS. The MS/MS spectra were initially analyzed by searching the human International Protein Index database using the SEQUEST software (1). Subsequently, new statistical algorithms were applied to the collective SEQUEST search results of each experiment. First, the PeptideProphet software (2) was applied to discriminate true assignments of MS/MS spectra to peptide sequences from false assignments, to assign a probability value for each identified peptide, and to compute the sensitivity and error rate for the assignment of spectra to sequences in each experiment. Second, the ProteinProphet software (3) was used to infer the protein identifications and to compute probabilities that a protein had been correctly identified, based on the available peptide sequence evidence. The resulting protein lists were filtered by a ProteinProphet probability score p > or = 0.5, which corresponded to an error rate of less than 5%. A total of 1,296, 1,430, and 1,476 proteins or related protein groups were identified in three subdatasets from the Huh7, HFH, and HH4 cells, respectively. In total, these subdatasets contained 2,486 unique protein identifications from human liver cells. An increase of the threshold to p > or = 0.9 (corresponding to an error rate of less than 1%) resulted in 2,159 unique protein identifications (1,146, 1,235, and 1,318 for the Huh7, HFH, and HH4 cells, respectively).     

Analysis of mammalian fatty acyl-coenzyme A species by mass spectrometry and tandem mass spectrometry

Acyl-CoAs are intermediates of numerous metabolic processes in eukaryotic cells, including beta-oxidation within mitochondria and peroxisomes, and the biosynthesis/remodeling of lipids (e.g. mono-, di-, and triglycerides, phospholipids and sphingolipids). Investigations of lipid metabolism have been advanced by the ability to quantitate acyl-CoA intermediates via liquid chromatography coupled to electrospray ionization-tandem mass spectrometric detection (LC-ESI-MS/MS), which is presently one of the most sensitive and specific analytical methods for both lipids and acyl-CoAs. This review of acyl-CoA analysis by mass spectrometry focuses on mammalian samples and long-chain analytes (i.e. palmitoyl-CoA), particularly reports of streamlined methodology, improved recovery, or expansion of the number of acyl chain-lengths amenable to quantitation.     

Assignment of disulfide bonds in proteins by partial acid hydrolysis and mass spectrometry

A new method is described for locating disulfide bonds in proteins which cannot be cleaved between half-cystinyl residues by enzymic methods, as is often the case for tightly coiled proteins, or for proteins in which half-cystinyl residues are not separated by residues required for enzymic cleavage. Partial acid hydrolysis of a model protein, hen egg-white lysozyme, produces a mixture of disulfide-containing peptides from which the disulfide connections may be deduced. The usefulness of a combination of HPLC, fast atom bombardment mass spectrometry, and computer-assisted analysis to identify disulfide-containing peptides present in the partial acid hydrolysate of the model protein is demonstrated. Chromatographic fractions of the hydrolysate were analyzed by mass spectrometry before and after chemical reduction of the disulfide bonds to determine the molecular weights of disulfide-containing peptides. Computer-assisted analysis was then used to relate the molecular weights of these peptides to specific segments of the protein from which the disulfide connectivities could be determined. Partial acid hydrolysis of proteins, which is attractive because it proceeds relatively independent of the amino acid sequence and structure, and because disulfide interchange is unlikely to occur in dilute acid, has become practical because disulfide-containing peptides present in complex mixtures can be identified rapidly and definitively by this method.     

Differential quantitative analysis of MHC ligands by mass spectrometry using stable isotope labeling

Currently, no method allows direct and quantitative comparison of MHC-presented peptides in pairs of samples, such as transfected and untransfected, tumorous and normal or infected and uninfected tissues or cell lines. Here we introduce two approaches that use isotopically labeled reagents to quantify by mass spectrometry the ratio of peptides from each source. The first method involves acetylation and is both fast and simple. However, higher peptide recoveries and a finer sensitivity are achieved by the second method, which combines guanidination and nicotinylation, because the charge state of peptides can be maintained. Using differential acetylation, we identified a beta catenin-derived peptide in solid colon carcinoma overpresented on human leucocyte antigen-A (HLA-A)(*)6801. Guanidination/nicotinylation was applied to keratin 18-transfected cells and resulted in the characterization of the peptide RLASYLDRV (HLA-A(*)0201), exclusively presented on the transfectant. Thus, we demonstrate methods that enable a pairwise quantitative comparison leading to the identification of overpresented MHC ligands.     

Human apolipoprotein A-I. Post-translational modification by fatty acid acylation

The human apolipoproteins are secretory proteins some of which have been shown to undergo proteolytic processing and post-translational addition of carbohydrate. Apolipoprotein A-I (apo-A-I), the predominant protein associated with high density lipoproteins, undergoes co-translational proteolytic processing as well as post-translational conversion of proapo-A-I to mature apo-A-I following cellular secretion. Utilizing the human hepatoma cell line HEP-G2, we have established that, in addition to proteolytic processing, secreted nascent apo-A-I is acylated with palmitate. Uniformly labeled [14C]palmitate and [1-14C]palmitate were each incorporated into apo-A-I when analyzed by sodium dodecyl sulfate gel electrophoresis and autoradiography. The acylation of apo-A-I with palmitate was confirmed by immunoprecipitation and gas chromatography/mass spectrometry. Hydroxylamine treatment resulted in the deacylation of apo-A-I. Although three of the apo-A-I isoforms analyzed by two-dimensional gel electrophoresis were shown to contain radio-labeled palmitate, 80% of acylated apo-A-I was in the proapolipoprotein A-I isoform. [14C]Oleate was not incorporated in secreted apo-A-I, indicating the specificity of the acylation of apo-A-I. Incubation of [14C] palmitate-acylated apo-A-I in serum and plasma under conditions in which proapo-A-I is proteolytically cleaved to mature apo-A-I did not result in deacylation. These data establish that fatty acid acylation occurs in human secretory proteins in addition to the previously reported acylation of cellular membrane proteins. These results suggest that the covalent linkage of lipids to apolipoproteins may play a critical role in apolipoprotein and lipoprotein metabolism.     

Lectin affinity capture,isotope-coded tagging and mass spectrometry to identify N-linked glycoproteins

We describe here a strategy for the large-scale identification of N-glycosylated proteins from a complex biological sample. The approach, termed isotope-coded glycosylation-site-specific tagging (IGOT), is based on the lectin column-mediated affinity capture of a set of glycopeptides generated by tryptic digestion of protein mixtures, followed by peptide-N-glycosidase-mediated incorporation of a stable isotope tag, 18O, specifically into the N-glycosylation site. The 18O-tagged peptides are then identified by multi-dimensional liquid chromatography-mass spectrometry (LC-MS)-based technology. The application of this method to the characterization of N-linked high-mannose and/or hybrid-type glycoproteins from an extract of Caenorhabditis elegans proteins allowed the identification of 250 glycoproteins, including 83 putative transmembrane proteins, with the simultaneous determination of 400 unique N-glycosylation sites. Because the method is applicable to the systematic identification of a wide range of glycoproteins, it should facilitate basic glycobiology research and may be useful for diagnostic applications, such as genome-wide screening for disease-related glycoproteins.     

Organelle proteomics of rat synaptic proteins: correlation-profiling by isotope-coded affinity tagging in conjunction with liquid chromatography-tandem mass spectrometry to reveal post-synaptic density specific proteins

Organelle proteomics is the method of choice for global analysis of cellular proteins. However, it is difficult to isolate organelles to homogeneity. Recently, correlation-profiling has been used to filter off the contaminants ad hoc and to disclose the genuine organelle-specific proteins. In the present study, we further extend the method to include subcellular compartments that contain proteins shared by multiple distinct subcellular domains. We performed correlation profiling of proteins contained in synaptic membrane and postsynaptic density (PSD) fractions isolated from rat brain. Proteins were labeled with isotope-coded affinity-tag reagents, digested with trypsin, and resulting peptides were resolved by cation exchange chromatography followed by reversed phase chromatography. Peptides were then subjected to mass spectrometry for quantification and identification. We confirm that the core PSD proteins were enriched in the PSD preparation. Other functional protein groups such as cytoskeleton-associated proteins, protein kinases and phosphatases, signaling components and regulators, as well as proteins involved in energy production partitioned to multiple organelles. When analyzed as groups, they were shown to accumulate to a lesser extent. Mitochondrial proteins and transporters were generally strongly depleted from the PSD fraction confirming that they were contaminants of the PSD preparation. Finally, immunoelectron microscopy was performed on selected proteins to validate the proteomics results, and confirm that synaptophysin that was highly depleted in the PSD preparation is localized in the presynaptic compartment, whereas LASP-1 that was slightly enriched in the PSD preparation is present in the PSD as well as other subdomains within the synapse.     

Site-specific phosphorylation profiling of Arabidopsis proteins by mass spectrometry and peptide chip analysis

An estimated one-third of all proteins in higher eukaryotes are regulated by phosphorylation by protein kinases (PKs). Although plant genomes encode more than 1000 PKs, the substrates of only a small fraction of these kinases are known. By mass spectrometry of peptides from cytoplasmic- and nuclear-enriched fractions, we determined 303 in vivo phosphorylation sites in Arabidopsis proteins. Among 21 different PKs, 12 were phosphorylated in their activation loops, suggesting that they were in their active state. Immunoblotting and mutational analysis confirmed a tyrosine phosphorylation site in the activation loop of a GSK3/shaggy-like kinase. Analysis of phosphorylation motifs in the substrates suggested links between several of these PKs and many target sites. To perform quantitative phosphorylation analysis, peptide arrays were generated with peptides corresponding to in vivo phosphorylation sites. These peptide chips were used for kinome profiling of subcellular fractions as well as H 2O 2-treated Arabidopsis cells. Different peptide phosphorylation profiles indicated the presence of overlapping but distinct PK activities in cytosolic and nuclear compartments. Among different H 2O 2-induced PK targets, a peptide of the serine/arginine-rich (SR) splicing factor SCL30 was most strongly affected. SRPK4 (SR protein-specific kinase 4) and MAPKs (mitogen-activated PKs) were found to phosphorylate this peptide, as well as full-length SCL30. However, whereas SRPK4 was constitutively active, MAPKs were activated by H 2O 2. These results suggest that SCL30 is targeted by different PKs. Together, our data demonstrate that a combination of mass spectrometry with peptide chip phosphorylation profiling has a great potential to unravel phosphoproteome dynamics and to identify PK substrates.     

Stable isotope-coded quaternization for comparative quantification of estrogen metabolites by high-performance liquid chromatography-electrospray ionization mass spectrometry

A fast and sensitive LC-ESI-MS method is described for the comparative quantification of 16 estrogen metabolites based on the derivatization of estrogens with a novel derivatizing reagent, N-methyl-nicotinic acid N-hydroxysuccinimide ester (C1-NA-NHS). The process introduces a quaternary amine to the analytes, making the analytes permanently charged regardless of the pH of the high-performance liquid chromatography (HPLC) mobile phase. This quaternization resulted in a highly efficient separation of 16 estrogen metabolites in 7 min at a detection level below 1 ng/mL. By using a deuterated derivatizing reagent (C1-d(3)-NA-NHS), a complete set of deuterated standards was utilized and used as internal standards in a comparative quantification and recovery study, demonstrating acceptable results over a wide concentration range. A pooled breast cancer serum sample was analyzed using the described method, and 15 estrogens were detected in the range of 80-530 pg/mL.     

Method for the quantitative assay of fatty acid-bile acid conjugates by tandem mass spectrometry

Fatty acid-bile acid conjugates and especially arachidyl amido cholic acid are synthetic molecules that were shown to prevent cholesterol gallstone formation in mice and hamsters as well as to dissolve pre-existing gallstones in mice. To measure these novel compounds we developed a liquid chromatography electrospray tandem mass spectrometry method based on the analysis of 100 microL of plasma with stearyl amido cholic acid (stamchol, 1.5 microM/L) added as internal standard. Repeatable calibrations between 0 and 50 microM/L exhibited consistent linearity and reproducibility. Inter- and intraassay C.V.s were 5.3-11.4% and 2.6-6.4%, respectively, at targeted concentrations of 0.1, 2.3 and 50 microM/L.     

Characterization of gel-separated glycoproteins using two-step proteolytic digestion combined with sequential microcolumns and mass spectrometry

Protein glycosylation can be vital for changing the function or physiochemical properties of a protein. Abnormal glycosylation can lead to protein malfunction, resulting in severe diseases. Therefore, it is important to develop techniques for characterization of such modifications in proteins at a sensitivity level comparable with state-of-the-art proteomics. Whereas techniques exist for characterization of high abundance glycoproteins, no single method is presently capable of providing information on both site occupancy and glycan structure on a single band excised from an electrophoretic gel. We present a new technique that allows characterization of low amounts of glycoproteins separated by gel electrophoresis. The method takes advantage of sequential specific and nonspecific enzymatic treatment followed by selective purification and characterization of the glycopeptides using graphite powder microcolumns in combination with mass spectrometry. The method is faster and more sensitive than previous approaches and is compatible with proteomic studies.     

Analysis of phosphorylated proteins and peptides by mass spectrometry

Phosphorylation on serine, threonine and tyrosine residues is an extremely important modulator of protein function. Therefore, there is a great need for methods capable of accurately elucidating sites of phosphorylation. Although full characterization of phosphoproteins remains a formidable analytical challenge, mass spectrometry has emerged as an increasingly viable tool for this task. This review summarizes the methodologies currently available for the analysis of phosphoproteins by mass spectrometry, including enrichment of compounds of interest using immobilized metal affinity chromatography and chemical tagging techniques, detection of phosphopeptides using mass mapping and precursor ion scans, localization of phosphorylation sites by peptide sequencing, and quantitation of phosphorylation by the introduction of mass tags. Despite the variety of powerful analytical methods that are now available, complete characterization of the phosphorylation state of a protein isolated in small quantities from a biological sample remains far from routine.