Analysis of the Arabidopsis cytosolic ribosome proteome provides detailed insights into its components and their post-translational modification

Finding gene-specific peptides by mass spectrometry analysis to pinpoint gene loci responsible for particular protein products is a major challenge in proteomics especially in highly conserved gene families in higher eukaryotes. We used a combination of in silico approaches coupled to mass spectrometry analysis to advance the proteomics insight into Arabidopsis cytosolic ribosomal composition and its post-translational modifications. In silico digestion of all 409 ribosomal protein sequences in Arabidopsis defined the proportion of theoretical gene-specific peptides for each gene family and highlighted the need for low m/z cutoffs of MS ion selection for MS/MS to characterize low molecular weight, highly basic ribosomal proteins. We undertook an extensive MS/MS survey of the cytosolic ribosome using trypsin and, when required, chymotrypsin and pepsin. We then used custom software to extract and filter peptide match information from Mascot result files and implement high confidence criteria for calling gene-specific identifications based on the highest quality unambiguous spectra matching exclusively to certain in silico predicted gene- or gene family-specific peptides. This provided an in-depth analysis of the protein composition based on 1446 high quality MS/MS spectra matching to 795 peptide sequences from ribosomal proteins. These identified peptides from five gene families of ribosomal proteins not identified previously, providing experimental data on 79 of the 80 different types of ribosomal subunits. We provide strong evidence for gene-specific identification of 87 different ribosomal proteins from these 79 families. We also provide new information on 30 specific sites of co- and post-translational modification of ribosomal proteins in Arabidopsis by initiator methionine removal, N-terminal acetylation, N-terminal methylation, lysine N-methylation, and phosphorylation. These site-specific modification data provide a wealth of resources for further assessment of the role of ribosome modification in influencing translation in Arabidopsis.     

Multiple covalent modifications of Trg, a sensory transducer of Escherichia coli

The sensory transducers of Escherichia coli are integral membrane proteins that mediate the tactic response of cells to chemical stimuli. Adaptation to environmental stimuli is correlated with methylation of the transducer proteins. Two transducer genes, tsr and tar, exhibit extensive homologies while no homology has been detected between a third transducer, trg, and those genes. The Tsr and Tar proteins have been shown to contain multiple sites for methylation as well as two sites for another modification that requires an active cheB gene product and is designated the CheB-dependent modification. In this study, covalent modifications of the Trg protein were characterized by analysis of tryptic peptides. We found that methylation occurred at several sites on the Trg protein and that the protein contained at least three sites for CheB-dependent modification, two of which were located on a tryptic peptide that contains both methionine and lysine. This tryptic peptide is analogous to the methionine- and lysine-containing methyl-accepting peptides isolated from the Tsr and Tar proteins and like those peptides may contain several methyl-accepting sites. We estimated the pKa of the group created by the CheB-dependent modification on the methionine- and lysine-containing peptide of Trg to be between pH 2.2 and 5.8. This result supports the idea that the CheB-dependent modification is an enzymatic deamidation of glutamine to glutamic acid.     

Strategies for the identification of arginine ADP-ribosylation sites

Mono-ADP-ribosylation of arginine is a protein modification in eukaryotic cells regulating protein activity and thereby influencing signal transduction and metabolism. Due to the complexity of the modification and the fragmentation pattern in MS/MS CID experiments, the identification of ADP-ribosylation sites in complex mixtures is difficult. Here we describe a two-step strategy, in the first step enriching and identifying potentially ADP-ribosylated proteins and in the second step identifying the sites of modification by a combination of LC/MS-, LC/MS(E) (MS at elevated fragmentation energy)- and LC/MS/MS experiments. Using this technique we could identify two ADP-ribosylation sites in TNFα digested with trypsin, protease V8 and both proteases and thereby demonstrate the specific ADP-ribosylation of TNFα. In complex samples the detection of ADP-ribosylated peptides requires further enrichment of the modified peptides. We tested various materials routinely used for the isolation of phosphopeptides. IMAC as well as TiO(2) chromatography were successfully applied for the selective enrichment of ADP-ribosylated model peptides.     

O-linked N-acetylglucosamine proteomics of postsynaptic density preparations using lectin weak affinity chromatography and mass spectrometry

O-GlcNAc is a widespread dynamic carbohydrate modification of cytosolic and nuclear proteins with features analogous to phosphorylation. O-GlcNAc acts critically in many cellular processes, including signal transduction, protein degradation, and regulation of gene expression. However, the study of its specific regulatory functions has been limited by difficulties in mapping sites of O-GlcNAc modification. We report methods for direct enrichment and identification of in vivo O-GlcNAc-modified peptides through lectin weak affinity chromatography (LWAC) and mass spectrometry. The effectiveness of this strategy on complex peptide mixtures was demonstrated through enrichment of 145 unique O-GlcNAc-modified peptides from a postsynaptic density preparation. 65 of these O-GlcNAc-modified peptides were sequenced and belonged to proteins with diverse functions in synaptic transmission. Beta-elimination/Michael addition, MS(3) on O-GlcNAc neutral loss ions, and electron capture dissociation were shown to facilitate analysis of O-GlcNAc-modified peptides/sites from lectin weak affinity chromatography enriched postsynaptic density samples. Bassoon and Piccolo, proteins critical to synapse assembly and vesicle docking, were extensively modified by O-GlcNAc. In some cases, O-GlcNAc was mapped to peptides previously identified as phosphorylated, indicating potential interplay between these modifications. Shared substrate amino acid context was apparent in subsets of O-GlcNAc-modified peptides, including "PVST" and a novel "TTA" motif (two hydroxyl-containing amino acids adjacent to an alanine). The results suggest specific roles for O-GlcNAc modification in synaptic transmission, establish a basis for site-specific regulatory studies, and provide methods that will facilitate O-GlcNAc proteome analysis across a wide variety of cells and tissues.     

Different methylation characteristics of protein arginine methyltransferase 1 and 3 toward the Ewing Sarcoma protein and a peptide

The multifunctional Ewing Sarcoma (EWS) protein, a member of a large family of RNA-binding proteins, is extensively asymmetrically dimethylated at arginine residues within RGG consensus sequences. Using recombinant proteins we examined whether type I protein arginine methyltransferase (PRMT)1 or 3 is responsible for asymmetric dimethylations of the EWS protein. After in vitro methylation of the EWS protein by GST-PRMT1, we identified 27 dimethylated arginine residues out of 30 potential methylation sites by mass spectrometry-based techniques (MALDI-TOF MS and MS/MS). Thus, PRMT1 recognizes most if not all methylation sites of the EWS protein. With GST-PRMT3, however, only nine dimethylated arginines, located mainly in the C-terminal region of EWS protein, could be assigned, indicating that structural determinants prevent complete methylation. In contrary to previous reports this study also revealed that trypsin is able to cleave after methylated arginines. Pull-down experiments showed that endogenous EWS protein binds efficiently to GST-PRMT1 but less to GST-PRMT3, which is in accordance to the in vitro methylation results. Furthermore, methylation of a peptide containing different methylation sites revealed differences in the site selectivity as well as in the kinetic properties of GST-PRMT1 and GST-PRMT3. Kinetic differences due to an inhibition effect of the methylation inhibitor S-adenosyl-L-homocysteine could be excluded by determining the corresponding K(i) values of the two enzymes and the K(d) values for the methyl donor S-adenosyl-L-methionine. The study demonstrates the strength of MS-based methods for a qualitative and quantitative analysis of enzymic arginine methylation, a posttranslational modification that becomes more and more the object of investigations.     

A novel proteomics approach to identify SUMOylated proteins and their modification sites in human cells

The small ubiquitin-related modifier (SUMO) is a small group of proteins that are reversibly attached to protein substrates to modify their functions. The large scale identification of protein SUMOylation and their modification sites in mammalian cells represents a significant challenge because of the relatively small number of in vivo substrates and the dynamic nature of this modification. We report here a novel proteomics approach to selectively enrich and identify SUMO conjugates from human cells. We stably expressed different SUMO paralogs in HEK293 cells, each containing a His(6) tag and a strategically located tryptic cleavage site at the C terminus to facilitate the recovery and identification of SUMOylated peptides by affinity enrichment and mass spectrometry. Tryptic peptides with short SUMO remnants offer significant advantages in large scale SUMOylome experiments including the generation of paralog-specific fragment ions following CID and ETD activation, and the identification of modified peptides using conventional database search engines such as Mascot. We identified 205 unique protein substrates together with 17 precise SUMOylation sites present in 12 SUMO protein conjugates including three new sites (Lys-380, Lys-400, and Lys-497) on the protein promyelocytic leukemia. Label-free quantitative proteomics analyses on purified nuclear extracts from untreated and arsenic trioxide-treated cells revealed that all identified SUMOylated sites of promyelocytic leukemia were differentially SUMOylated upon stimulation.     

Identification of Novel Site‐Specific Alterations in the Modification Level of Myelin Basic Protein Isolated from Mouse Brain at Different Ages Using Capillary Electrophoresis–Mass Spectrometry

Myelin basic protein (MBP) is a multifunctional protein involved in maintaining the stability and integrity of the myelin sheath by a variety of interactions with membranes and other proteins. MBP is subjected to extensive posttranslational modifications (PTMs) that are known to be crucial for the regulation of these interactions. Here, we report capillary electrophoresis–mass spectrometric (CE–MS) analysis for the separation and identification of MBP peptides that incorporate the same PTM at different sites, creating multiple localization variants, and the ability to analyze challenging modifications such as asparagine and glutamine deamidation, isomerization, and arginine citrullination. Moreover, we observed site‐specific alterations in the modification level of MBP purified from brain of mice of different age. In total, we identified 40 modifications at 33 different sites, which include both previously reported and seven novel modifications. The identified modifications include Nα‐terminal acetylation, mono‐ and dimethylation, phosphorylation, oxidation, deamidation, and citrullination. Notably, some new sites of arginine methylation overlap with the sites of citrullination. Our results highlight the need for sensitive and efficient techniques for a comprehensive analysis of PTMs.     

Identification and characterization of phosphorylated proteins in the human pituitary

Post-translational modifications of proteins from the human pituitary gland play an important role in the regulation of different body functions. We report on the application of a liquid chromatography-tandem mass spectrometry (MS/MS) based approach to detect and characterize phosphorylated proteins in a whole human pituitary digest. By combining an immobilized metal affinity column-based enrichment method with MS/MS conditions that favor the neutral loss of phosphoric acid from a phosphorylated precursor ion, we identified several previously undescribed phosphorylated peptides. The identified peptides were matched to the sequences of six pituitary proteins: the human growth hormone, chromogranin A, secretogranin I, 60S ribosomal protein P1 and/or P2, DnaJ homolog subfamily C member 5, and galanin. The phosphorylation sites of these important regulatory proteins were determined by MS/MS and MS(3) analysis.     

Characterisation of cisplatin coordination sites in cellular <Emphasis Type="Italic">Escherichia coli</Emphasis> DNA-binding proteins by combined biphasic liquid chromatography and ESI tandem mass spectrometry

Combined multidimensional liquid chromatography and electrospray ionisation tandem mass spectrometry was employed to analyse platinated tryptic peptides from Escherichia coli cells treated with the anticancer drug cis-[PtCl₂(NH₃)₂] at pH 7.0. Prerequisites for the LC/LC/MS/MS analysis of protein targets that are fulfilled by cisplatin are (a) that the original protein binding sites have a high kinetic stability over the range 2.3 < pH < 8.5, and (b) that the metal fragment remains coordinated to a significant number of b⁺ and y⁺ peptide ions under MS/MS fragmentation conditions. Matching the MS/MS spectra of the platinated tryptic peptides to sequences of proteins in the E. coli database enabled the identification of 31 protein targets for cisplatin. Whereas six of these are high-abundance enzymes and ribosomal proteins in E. coli cells, five low-abundance DNA-binding proteins were also identified as specific targets. These include the DNA mismatch repair protein mutS, the DNA helicase II (uvrD) and topoisomerase I (top1). Two efflux proteins (acrD, mdtA), the redox regulator thioredoxin 1 (thiO) and the external filament-like type-1 fimbrial protein A chain (fimA1) were also characterised as specific cisplatin-binding proteins. Kinetically favoured carboxylate (D, E) and hydroxy (S, T, Y) O atoms were identified as the Pt coordination sites in 18 proteins and methionyl S atoms in 9 proteins.     

Tryptic digestion of ubiquitin standards reveals an improved strategy for identifying ubiquitinated proteins by mass spectrometry

Ubiquitination plays an essential role in maintaining cellular homeostasis by regulating a multitude of essential processes. The ability to identify ubiquitinated proteins by MS currently relies on a strategy in which ubiquitinated peptides are identified by a 114.1 Da diglycine (GG) tag on lysine residues, which is derived from the C-terminus of ubiquitin, following trypsin digestion. In the following study, we report a more comprehensive approach for mapping ubiquitination sites by trypsin digestion and MS/MS analysis. We demonstrate that ubiquitination sites can be identified by signature peptides containing a GG-tag (114.1 Da) and an LRGG-tag (383.2 Da) on internal lysine residues as well as a GG-tag found on the C-terminus of ubiquitinated peptides. Application of this MS-based approach enabled the identification of 96 ubiquitination sites from proteins purified from human MCF-7 breast cancer cells, representing a 2.4-fold increase in the number of ubiquitination sites that could be identified over standard methods. Our improved MS-based strategy will aid future studies which aim to identify and/or characterize ubiquitinated proteins in human cells.     

Characterization of the mouse pancreatic islet proteome and comparative analysis with other mouse tissues

The pancreatic islets of Langerhans, and especially the insulin-producing beta cells, play a central role in the maintenance of glucose homeostasis. Alterations in the expression of multiple proteins in the islets that contribute to the maintenance of islet function are likely to underlie the pathogenesis of types 1 and 2 diabetes. To identify proteins that constitute the islet proteome, we provide the first comprehensive proteomic characterization of pancreatic islets for mouse, the most commonly used animal model in diabetes research. Using strong cation exchange fractionation coupled with reversed phase LC-MS/MS we report the confident identification of 17,350 different tryptic peptides covering 2612 proteins having at least two unique peptides per protein. The data set also identified approximately 60 post-translationally modified peptides including oxidative modifications and phosphorylation. While many of the identified phosphorylation sites corroborate those previously known, the oxidative modifications observed on cysteinyl residues reveal potentially novel information suggesting a role for oxidative stress in islet function. Comparative analysis with 15 available proteomic data sets from other mouse tissues and cells revealed a set of 133 proteins predominantly expressed in pancreatic islets. This unique set of proteins, in addition to those with known functions such as peptide hormones secreted from the islets, contains several proteins with as yet unknown functions. The mouse islet protein and peptide database accessible at (http://ncrr.pnl.gov), provides an important reference resource for the research community to facilitate research in the diabetes and metabolism fields.     

Identification of phosphorylation sites on phosducin-like protein by QTOF mass spectrometry.

Post-translational modifications are used by cells to control the functions of proteins. Phosducin-like protein (PhLP) is a regulator of G-protein signaling that is post-translationally modified via phosphorylation. Phosphorylation of PhLP initiates its degradation by the 26S proteasome in serum-stimulated cells. In this report, we show that PhLP is phosphorylated in serum-stimulated Chinese hamster ovary (CHO) cells. Through the use of tandem mass spectrometry (MS/MS), the specific amino acids phosphorylated can be identified. A PhLP-myc-His construct was purified and phosphorylated by serum-stimulated CHO extract. The resulting protein was digested with trypsin and the peptides were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Automated collison-induced dissociation data acquisition was compared with LC-MS/MS of manually chosen parents. In general, LC-MS/MS is superior for parent ions chosen manually, with the notable exception that automated fragmentation employs dynamic collision energy, which can result in higher quality collison-induced dissociation. Using the LC-MS/MS methods, four phosphorylation sites on PhLP were positively identified.     

Identification of Cep169-interacting proteins and the in vivo modification sites of Cep169 via proteomic analysis

Cep169 is a microtubule plus-end tracking and centrosomal protein that interacts with CDK5RAP2. Cep169 is known to regulate microtubule dynamics and stability; however, its other cellular functions remain largely elusive. In this study, we identified novel Cep169-interacting proteins from HeLa cell extracts. Proteomic analysis via LC-MS/MS helped to identify approximately 400 novel Cep169-interacting proteins, including centrosomal proteins, cilium proteins, microtubule-associating proteins, and several E3 ubiquitin ligases. In addition, we identified in vivo posttranslational modification sites of Cep169, namely, 27 phosphorylation sites, five methylation sites, and four ubiquitination sites. Of these, 14 phosphorylated residues corresponding to the consensus Cdk phosphorylation sites may be required for Cdk1-mediated dissociation of Cep169 from the centrosome during mitosis and Cdk regulation during the G1/S phase. Furthermore, siRNA-induced Cep169 depletion was found to inhibit the growth of RPE1 cells. Our findings suggest that Cep169 regulates cell growth by interacting with multiple proteins.     

Proteome-wide profiling of carbonylated proteins and carbonylation sites in HeLa cells under mild oxidative stress conditions

A number of oxidative protein modifications have been well characterized during the past decade. Presumably, reversible oxidative posttranslational modifications (PTMs) play a significant role in redox signaling pathways, whereas irreversible modifications including reactive protein carbonyl groups are harmful, as their levels are typically increased during aging and in certain diseases. Despite compelling evidence linking protein carbonylation to numerous disorders, the underlying molecular mechanisms at the proteome remain to be identified. Recent advancements in analysis of PTMs by mass spectrometry provided new insights into the mechanisms of protein carbonylation, such as protein susceptibility and exact modification sites, but only for a limited number of proteins. Here we report the first proteome-wide study of carbonylated proteins including modification sites in HeLa cells for mild oxidative stress conditions. The analysis relied on our recent strategy utilizing mass spectrometry-based enrichment of carbonylated peptides after DNPH derivatization. Thus a total of 210 carbonylated proteins containing 643 carbonylation sites were consistently identified in three replicates. Most carbonylation sites (284, 44.2%) resulted from oxidation of lysine residues (aminoadipic semialdehyde). Additionally, 121 arginine (18.8%), 121 threonine (18.8%), and 117 proline residues (18.2%) were oxidized to reactive carbonyls. The sequence motifs were significantly enriched for lysine and arginine residues near carbonylation sites (±10 residues). Gene Ontology analysis revealed that 80% of the carbonylated proteins originated from organelles, 50% enrichment of which was demonstrated for the nucleus. Moreover, functional interactions between carbonylated proteins of kinetochore/spindle machinery and centrosome organization were significantly enriched. One-third of the 210 carbonylated proteins identified here are regulated during apoptosis.     

Quantitative phosphoproteome analysis using a dendrimer conjugation chemistry and tandem mass spectrometry

We present a robust and general method for the identification and relative quantification of phosphorylation sites in complex protein mixtures. It is based on a new chemical derivatization strategy using a dendrimer as a soluble polymer support and tandem mass spectrometry (MS/MS). In a single step, phosphorylated peptides are covalently conjugated to a dendrimer in a reaction catalyzed by carbodiimide and imidazole. Modified phosphopeptides are released from the dendrimer via acid hydrolysis and analyzed by MS/MS. When coupled with an initial antiphosphotyrosine protein immunoprecipitation step and stable-isotope labeling, in a single experiment, we identified all known tyrosine phosphorylation sites within the immunoreceptor tyrosine-based activation motifs (ITAM) of the T-cell receptor (TCR) CD3 chains, and previously unknown phosphorylation sites on total 97 tyrosine phosphoproteins and their interacting partners in human T cells. The dynamic changes in phosphorylation were quantified in these proteins.     

Elucidation of N-glycosylation sites on human platelet proteins: a glycoproteomic approach

Among known platelet proteins, a prominent and functionally important group is represented by glycoprotein isoforms. They account e.g. for secretory proteins and plasma membrane receptors including integrins and glycoprotein VI as well as intracellular components of cytosol and organelles including storage proteins (multimerin 1 etc.). Although many of those proteins have been studied for some time with regard to their function, little attention has been paid with respect to their glycosylation sites. Here we report the analysis of N-glycosylation sites of human platelet proteins. For the enrichment of glycopeptides, lectin affinity chromatography as well as chemical trapping of protein bound oligosaccharides was used. Therefore, concanavalin A was used for specific interaction with carbohydrate species along with periodic acid oxidation and hydrazide bead trapping of glycosylated proteins. Derivatization by peptide:N-glycosidase F yielded deglycosylated peptides, which provided the basis for the elucidation of proteins and their sites of modification. Using both methods in combination with nano-LC-ESI-MS/MS analysis 70 different glycosylation sites within 41 different proteins were identified. Comparison with the Swiss-Prot database established that the majority of these 70 sites have not been specifically determined by previous research projects. With this approach including hydrazide bead affinity trapping, the immunoglobulin receptor G6f, which is known to couple to the Ras-mitogen-activated protein kinase pathway in the immune system, was shown here for the first time to be present in human platelets.     

An antibody-free enrichment approach enabled by reductive glutaraldehydation for monomethyllysine proteome analysis

Protein lysine monomethylation is an important post-translational modification participated in regulating many biological processes. There is growing interest in identifying these methylation events. However, the introduction of one methyl group on lysine residues has negligible effect on changing the physical and chemical properties of proteins or peptides, making enriching and identifying monomethylated lysine (Kme1) proteins or peptides extraordinarily challenging. In this study, we proposed an antibody-free chemical proteomics approach to capture Kme1 peptides from complex protein digest. By exploiting reductive glutaraldehydation, 5-aldehyde-pentanyl modified Kme1 residues and piperidine modified primary amines were generated at the same time. The peptides with aldehyde modified Kme1 residues were then enriched by solid-phase hydrazide chemistry. This chemical proteomics approach was validated by using several synthetic peptides. It was demonstrated that it can enrich and detect Kme1 peptide from peptide mixture containing 5000-fold more bovine serum albumin tryptic digest. Besides, we extended our approach to profile Kme1 using heavy methyl stable isotope labeling by amino acids in cell culture (hmSILAC) labeled Jurkat T cells and Hela cells. Totally, 29 Kme1 sites on 25 proteins were identified with high confidence and 11 Kme1 sites were identified in both two types cells. This is the first antibody-free chemical proteomics approach to enrich Kme1 peptides from complex protein digest, and it provides a potential avenue for the analysis of methylome.     

Identification and quantification of N-linked glycoproteins using hydrazide chemistry,stable isotope labeling and mass spectrometry

Quantitative proteome profiling using stable isotope protein tagging and automated tandem mass spectrometry (MS/MS) is an emerging technology with great potential for the functional analysis of biological systems and for the detection of clinical diagnostic or prognostic marker proteins. Owing to the enormous complexity of proteomes, their comprehensive analysis is an as-yet-unresolved technical challenge. However, biologically or clinically important information can be obtained if specific, information-rich protein classes, or sub-proteomes, are isolated and analyzed. Glycosylation is the most common post-translational modification. Here we describe a method for the selective isolation, identification and quantification of peptides that contain N-linked carbohydrates. It is based on the conjugation of glycoproteins to a solid support using hydrazide chemistry, stable isotope labeling of glycopeptides and the specific release of formerly N-linked glycosylated peptides via peptide- N-glycosidase F (PNGase F). The recovered peptides are then identified and quantified by MS/MS. We applied the approach to the analysis of plasma membrane proteins and proteins contained in human blood serum.