Defining the filarial N-glycoproteome by glycosite mapping in the human parasitic nematode Brugia malayi.

N-linked glycosylation is a critical post translational modification of eukaryotic proteins. N-linked glycans are present on surface and secreted filarial proteins that play a role in host parasite interactions. Examples of glycosylated Brugia malayi proteins have been previously identified but there has not been a systematic study of the N-linked glycoproteome of this or any other filarial parasite. In this study, we applied an enhanced N-glyco FASP protocol using an engineered carbohydrate-binding protein, Fbs1, to enrich N-glycosylated peptides for analysis by LC-MS/MS. We then mapped the N-glycosites on proteins from three host stages of the parasite: adult female, adult male and microfilariae. Fbs1 enrichment of N-glycosylated peptides enhanced the identification of N-glycosites. Our data identified 582 N-linked glycoproteins with 1273 N-glycosites. Gene ontology and cell localization prediction of the identified N-glycoproteins indicated that they were mostly membrane and extracellular proteins. Comparing results from adult female worms, adult male worms, and microfilariae, we find variability in N-glycosylation at the protein level as well as at the individual N-glycosite level. These variations are highlighted in cuticle N-glycoproteins and adult worm restricted N-glycoproteins as examples of proteins at the host parasite interface that are well positioned as potential therapeutic targets or biomarkers.

Polyglutamylation: biology and analysis.

Polyglutamylation is a posttranslational modification (PTM) that adds several glutamates on glutamate residues in the form of conjugated peptide chains by a family of enzymes known as polyglutamylases. Polyglutamylation is well documented in microtubules. Polyglutamylated microtubules consist of different α- and ß-tubulin subunits with varied number of added glutamate residues. Kinetic control and catalytic rates of tubulin modification by polyglutamylases influence the polyglutamylation pattern of functional microtubules. The recent studies uncovered catalytic mechanisms of the glutamylation enzymes family, particularly tubulin tyrosine ligase-like (TTLL). Variable length polyglutamylation of primary sequence glutamyl residues have been mapped with a multitude of protein chemistry and proteomics approaches. Although polyglutamylation was initially considered a tubulin-specific modification, the recent studies have uncovered a calmodulin-dependent glutamylase, SidJ. Nano-electrospray ionization (ESI) proteomic approaches have identified quantifiable polyglutamylated sites in specific substrates. Indeed, conjugated glutamylated peptides were used in nano-liquid chromatography gradient delivery due to their relative hydrophobicity for their tandem mass spectrometry (MS/MS) characterization. The recent polyglutamylation characterization has revealed three major sites: E445 in α-tubulin, E435 in ß-tubulin, and E860 in SdeA. In this review, we have summarized the progress made using proteomic approaches for large-scale detection of polyglutamylated peptides, including biology and analysis.

A bi-specific lectin from the mushroom Boletopsis grisea and its application in glycoanalytical workflows.

The BLL lectin from the edible Japanese "Kurokawa" mushroom (Boletopsis leucomelaena) was previously reported to bind to N-glycans harboring terminal N-acetylglucosamine (GlcNAc) and to induce apoptosis in a leukemia cell line. However, its gene has not been reported. In this study, we used a transcriptomics-based workflow to identify a full-length transcript of a BLL functional ortholog (termed BGL) from Boletopsis grisea, a close North American relative of B. leucomelaena. The deduced amino acid sequence of BGL was an obvious member of fungal fruit body lectin family (Pfam PF07367), a highly conserved group of mushroom lectins with a preference for binding O-glycans harboring the Thomsen-Friedenreich antigen (TF-antigen; Galß1,3GalNAc-α-) and having two ligand binding sites. Functional characterization of recombinant BGL using glycan microarray analysis and surface plasmon resonance confirmed its ability to bind both the TF-antigen and ß-GlcNAc-terminated N-glycans. Structure-guided mutagenesis of BGL's two ligand binding clefts showed that one site is responsible for binding TF-antigen structures associated with O-glycans, whereas the second site specifically recognizes N-glycans with terminal ß-GlcNAc. Additionally, the two sites show no evidence of allosteric communication. Finally, mutant BGL proteins having single functional bindings site were used to enrich GlcNAc-capped N-glycans or mucin type O-glycopeptides from complex samples in glycomics and glycoproteomics analytical workflows.

Mapping of polyglutamylation in tubulins using nanoLC-ESI-MS/MS.

Tubulin polyglutamylation is a polymeric modification that extends from the carboxyl-terminus of tubulins. Molecular description of amino acids and their branching polyglutamyls is a hallmark of tubulin in microtubules. There are different chemical approaches for detecting these polymeric structures, mostly reported prior to development of nESI peptide analysis. Here we demonstrate a novel and simple approach to detect shared regions of amino acid ions from tubulin polyglutamylated peptides in nanoLC-MS/MS. This involves two parallel in gel digestions with trypsin and subtilisin followed by mapping of di- and triglutamyl modifications of α- and ß-tubulins using a routine proteomics assay. We present three levels of information: i) identification of proteomics MS/MS data, ii) description of internal fragment ion series common across digests, and iii) extracted ion chromatograms mapped relative to retention time standards for confirmation of relative hydrophobicity values. Our nanoLC assay positive ion ESI detects up to 3 conjugated glutamates in tubulins. We implemented an analytical column only bottom up approach that characterizes molecular features of polyglutamylated tubulins.

Computational and experimental analysis of the glycophosphatidylinositol-anchored proteome of the human parasitic nematode Brugia malayi.

Further characterization of essential systems in the parasitic filarial nematode Brugia malayi is needed to better understand its biology, its interaction with its hosts, and to identify critical components that can be exploited to develop novel treatments. The production of glycophosphatidylinositol-anchored proteins (GPI-APs) is essential for eukaryotic cellular and physiological function. In addition, GPI-APs perform many important roles for cells. In this study, we characterized the B. malayi GPI-anchored proteome using both computational and experimental approaches. We used bioinformatic strategies to show the presence or absence of B. malayi GPI-AP biosynthetic pathway genes and to compile a putative B. malayi GPI-AP proteome using available prediction programs. We verified these in silico analyses using proteomics to identify GPI-AP candidates prepared from the surface of intact worms and from membrane enriched extracts. Our study represents the first description of the GPI-anchored proteome in B. malayi and lays the groundwork for further exploration of this essential protein modification as a target for novel anthelmintic therapeutic strategies.

S-adenosylhomocysteine Hydrolase Participates in DNA Methylation Inheritance.

DNA (cytosine-5) methyltransferase 1 (DNMT1) is essential for mammalian development and maintenance of DNA methylation following DNA replication in cells. The DNA methylation process generates S-adenosyl-l-homocysteine, a strong inhibitor of DNMT1. Here we report that S-adenosylhomocysteine hydrolase (SAHH/AHCY), the only mammalian enzyme capable of hydrolyzing S-adenosyl-l-homocysteine binds to DNMT1 during DNA replication. SAHH enhances DNMT1 activity in vitro, and its overexpression in mammalian cells led to hypermethylation of the genome, whereas its inhibition by adenosine periodate or siRNA-mediated knockdown resulted in hypomethylation of the genome. Hypermethylation was consistent in both gene bodies and repetitive DNA elements leading to aberrant gene regulation. Cells overexpressing SAHH specifically up-regulated metabolic pathway genes and down-regulated PPAR and MAPK signaling pathways genes. Therefore, we suggest that alteration of SAHH level affects global DNA methylation levels and gene expression.

An engineered high affinity Fbs1 carbohydrate binding protein for selective capture of N-glycans and N-glycopeptides.

A method for selective and comprehensive enrichment of N-linked glycopeptides was developed to facilitate detection of micro-heterogeneity of N-glycosylation. The method takes advantage of the inherent properties of Fbs1, which functions within the ubiquitin-mediated degradation system to recognize the common core pentasaccharide motif (Man3GlcNAc2) of N-linked glycoproteins. We show that Fbs1 is able to bind diverse types of N-linked glycomolecules; however, wild-type Fbs1 preferentially binds high-mannose-containing glycans. We identified Fbs1 variants through mutagenesis and plasmid display selection, which possess higher affinity and improved recovery of complex N-glycomolecules. In particular, we demonstrate that the Fbs1 GYR variant may be employed for substantially unbiased enrichment of N-linked glycopeptides from human serum. Most importantly, this highly efficient N-glycopeptide enrichment method enables the simultaneous determination of N-glycan composition and N-glycosites with a deeper coverage (compared to lectin enrichment) and improves large-scale N-glycoproteomics studies due to greatly reduced sample complexity.

A tool to evaluate correspondence between extraction ion chromatographic peaks and peptide-spectrum matches in shotgun proteomics experiments.

Chromatographed peptide signals form the basis of further data processing that eventually results in functional information derived from data-dependent bottom-up proteomics assays. We seek to rank LC/MS parent ions by the quality of their extracted ion chromatograms. Ranked extracted ion chromatograms act as an intuitive physical/chemical preselection filter to improve the quality of MS/MS fragment scans submitted for database search. We identify more than 4900 proteins when considering detector shifts of less than 7 ppm. High quality parent ions for which the database search yields no hits become candidates for subsequent unrestricted analysis for PTMs. Following this rational approach, we prioritize identification of more than 5000 spectrum matches from modified peptides and confirmed the presence of acetylaldehyde-modified His/Lys. We present a logical workflow that scores data-dependent selected ion chromatograms and leverage information about semianalytical LC/LC dimension prior to MS. Our method can be successfully used to identify unexpected modifications in peptides with excellent chromatography characteristics, independent of fragmentation pattern and activation methods. We illustrate analysis of ion chromatograms detected in two different modes by RF linear ion trap and electrostatic field orbitrap.

Proteomics by mass spectrometry: approaches, advances, and applications.

Mass spectrometry (MS) is the most comprehensive and versatile tool in large-scale proteomics. In this review, we dissect the overall framework of the MS experiment into its key components. We discuss the fundamentals of proteomic analyses as well as recent developments in the areas of separation methods, instrumentation, and overall experimental design. We highlight both the inherent strengths and limitations of protein MS and offer a rough guide for selecting an experimental design based on the goals of the analysis. We emphasize the versatility of the Orbitrap, a novel mass analyzer that features high resolution (up to 150,000), high mass accuracy (2-5 ppm), a mass-to-charge range of 6000, and a dynamic range greater than 10(3). High mass accuracy of the Orbitrap expands the arsenal of the data acquisition and analysis approaches compared with a low-resolution instrument. We discuss various chromatographic techniques, including multidimensional separation and ultra-performance liquid chromatography. Multidimensional protein identification technology (MudPIT) involves a continuum sample preparation, orthogonal separations, and MS and software solutions. We discuss several aspects of MudPIT applications to quantitative phosphoproteomics. MudPIT application to large-scale analysis of phosphoproteins includes (a) a fractionation procedure for motif-specific enrichment of phosphopeptides, (b) development of informatics tools for interrogation and validation of shotgun phosphopeptide data, and (c) in-depth data analysis for simultaneous determination of protein expression and phosphorylation levels, analog to western blot measurements. We illustrate MudPIT application to quantitative phosphoproteomics of the beta adrenergic pathway. We discuss several biological discoveries made via mass spectrometry pipelines with a focus on cell signaling proteomics.

Progressive aggregation despite chaperone associations of a mutant SOD1-YFP in transgenic mice that develop ALS.

Recent studies suggest that superoxide dismutase 1 (SOD1)-linked amyotrophic lateral sclerosis results from destabilization and misfolding of mutant forms of this abundant cytosolic enzyme. Here, we have tracked the expression and fate of a misfolding-prone human SOD1, G85R, fused to YFP, in a line of transgenic G85R SOD1-YFP mice. These mice, but not wild-type human SOD1-YFP transgenics, developed lethal paralyzing motor symptoms at 9 months. In situ RNA hybridization of spinal cords revealed predominant expression in motor neurons in spinal cord gray matter in all transgenic animals. Concordantly, G85R SOD-YFP was diffusely fluorescent in motor neurons of animals at 1 and 6 months of age, but at the time of symptoms, punctate aggregates were observed in cell bodies and processes. Biochemical analyses of spinal cord soluble extracts indicated that G85R SOD-YFP behaved as a misfolded monomer at all ages. It became progressively insoluble at 6 and 9 months of age, associated with presence of soluble oligomers observable by gel filtration. Immunoaffinity capture and mass spectrometry revealed association of G85R SOD-YFP, but not WT SOD-YFP, with the cytosolic chaperone Hsc70 at all ages. In addition, 3 Hsp110's, nucleotide exchange factors for Hsp70s, were captured at 6 and 9 months. Despite such chaperone interactions, G85R SOD-YFP formed insoluble inclusions at late times, containing predominantly intermediate filament proteins. We conclude that motor neurons, initially "compensated" to maintain the misfolded protein in a soluble state, become progressively unable to do so.

Colander: a probability-based support vector machine algorithm for automatic screening for CID spectra of phosphopeptides prior to database search.

We developed a probability-based machine-learning program, Colander, to identify tandem mass spectra that are highly likely to represent phosphopeptides prior to database search. We identified statistically significant diagnostic features of phosphopeptide tandem mass spectra based on ion trap CID MS/MS experiments. Statistics for the features are calculated from 376 validated phosphopeptide spectra and 376 nonphosphopeptide spectra. A probability-based support vector machine (SVM) program, Colander, was then trained on five selected features. Data sets were assembled both from LC/LC-MS/MS analyses of large-scale phosphopeptide enrichments from proteolyzed cells, tissues and synthetic phosphopeptides. These data sets were used to evaluate the capability of Colander to select pS/pT-containing phosphopeptide tandem mass spectra. When applied to unknown tandem mass spectra, Colander can routinely remove 80% of tandem mass spectra while retaining 95% of phosphopeptide tandem mass spectra. The program significantly reduced computational time spent on database search by 60-90%. Furthermore, prefiltering tandem mass spectra representing phosphopeptides can increase the number of phosphopeptide identifications under a predefined false positive rate.

Motif-specific sampling of phosphoproteomes.

Phosphoproteomics, the targeted study of a subfraction of the proteome which is modified by phosphorylation, has become an indispensable tool to study cell signaling dynamics. We described a methodology that linked phosphoproteome and proteome analysis based on Ba2+ binding properties of amino acids. This technology selected motif-specific phosphopeptides independent of the system under analysis. MudPIT (Multidimensional Identification Technology) identified 1037 precipitated phosphopeptides from as little as 250 microg of proteins. To extend coverage of the phosphoproteome, we sampled the nuclear extract of HeLa cells with three values of Ba2+ ions molarity. The presence of more than 70% of identified phosphoproteins was further substantiated by their nonmodified peptides. Upon isoproterenol stimulation of HEK cells, we identified an increasing number of phosphoproteins from MAPK cascades and AKAP signaling hubs. We quantified changes in both protein and phosphorylation levels of 197 phosphoproteins including a critical kinase, MAPK1. Integration of differential phosphorylation of MAPK1 with knowledge bases constructed modules that correlated well with its role as node in cross-talk of canonical pathways.

Anion and cation mixed-bed ion exchange for enhanced multidimensional separations of peptides and phosphopeptides.

Shotgun proteomics typically uses multidimensional LC/MS/MS analysis of enzymatically digested proteins, where strong cation-exchange (SCX) and reversed-phase (RP) separations are coupled to increase the separation power and dynamic range of analysis. Here we report an on-line multidimensional LC method using an anion- and cation-exchange mixed bed for the first separation dimension. The mixed-bed ion-exchange resin improved peptide recovery over SCX resins alone and showed better orthogonality to RP separations in two-dimensional separations. The Donnan effect, which was enhanced by the introduction of fixed opposite charges in one column, is proposed as the mechanism responsible for improved peptide recovery by producing higher fluxes of salt cations and lower populations of salt anions proximal to the SCX phase. An increase in orthogonality was achieved by a combination of increased retention for acidic peptides and moderately reduced retention of neutral to basic peptides by the added anion-exchange resin. The combination of these effects led to approximately 100% increase in the number of identified peptides from an analysis of a tryptic digest of a yeast whole cell lysate. The application of the method to phosphopeptide-enriched samples increased by 94% phosphopeptide identifications over SCX alone. The lower pKa of phosphopeptides led to specific enrichment in a single salt step resolving acidic phosphopeptides from other phospho- and non-phosphopeptides. Unlike previous methods that use anion exchange to alter selectivity or enrich phosphopeptides, the proposed format is unique in that it works with typical acidic buffer systems used in electrospray ionization, making it feasible for online multidimensional LC/MS/MS applications.

Automated ultra-high-pressure multidimensional protein identification technology (UHP-MudPIT) for improved peptide identification of proteomic samples.

Multidimensional separation is one of the most successful approaches for proteomics studies that deal with complex samples. We have developed an automated ultra-high-pressure multidimensional liquid chromatography system that operates up to approximately 20 kpsi to improve separations and increase protein coverage from limited amount of samples. The reversed-phase gradient is operated in the constant-flow mode opposed to the constant-pressure mode, which is typical of previous ultra-high-pressure systems. In contrast to constant-pressure systems, the gradient shape is fully controllable and can be optimized for the type of samples to be run. The system also features fast sample loading/desalting using a vented column approach to improve sample throughput. This approach was validated on a soluble fraction from yeast lysate where we achieved approximately 30% more protein identifications using a 60-cm-long triphasic capillary column than with our traditional approach. Advantages of the use of a relatively long reversed-phase column (approximately 50 cm) for MudPIT-type experiments are also discussed.

Intregrated analysis of the human cardiac transcriptome, proteome and phosphoproteome.

Altered expression of different classes of genes has been shown to differentiate between failing and nonfailing human hearts. However, characterization of proteins and the post-translational modifications that regulate their functions is required for understanding both the physiology of cardiac muscle and the mechanisms leading to pathological states associated with cardiac diseases. We present in this paper, an analysis of the human cardiac transcriptome, proteome and phosphoproteome. Data from two sources (i) experiments performed in our laboratory and (ii) bioinformatics searches of public databases (SWISS-PROT, NCBI, Cardiac Gene Expression Knowledge Base, Gene Ontology Consortium and Affymetrix) are reported in a relational database that allows user-designed specific queries. Microarray experiments were performed with Affymetrix Hu95Av2. Cardiac proteins were digested with trypsin. An 11 step cation exchange procedure produced fractions for analysis in separate reversed phase high-performance liquid chromatography-tandem mass spectrometry (MS/MS) experiments. Immobilized metal affinity chromatography was used to select the phosphopeptides from the same tryptic peptide mixture. They were then further investigated by MS/MS. Gel-free approaches were used to detect 267 proteins and 47 phosphopeptides. Our human cardiac database contains 447 entries. We propose the use of this platform, built with data derived from nonfailing hearts, as a template for initiating the effort to characterize the human cardiac proteome and its associated post-translational modifications.

Site-specific quantitation of protein nitration using liquid chromatography/tandem mass spectrometry.

The native reference peptide (NRP) method has been adapted to the measure of the degree of protein nitration at a specific tyrosine residue. In these experiments, human serum albumin was modified in a myeloperoxidase-mediated reaction in the presence of nitrite, with nitration detected predominantly at one site, Y162. The time-dependent increase in nitration at this site was measured based on the increasing abundance of the peptide 162YnLYEIAR168 and the corresponding decrease in the 162YLYEIAR168 peptide in in-gel trypsin digests. The peptide 66LVNEVTEFAK75, also formed in the tryptic digest, was used as the native reference peptide. Quantitation was achieved by determining the chromatographic peak area of the two analyte peptides relative to the native reference peptide by LC/tandem mass spectrometric analyses with selected reaction monitoring. The NRP results were validated by correlation to the time-dependent increase in total protein-nitrotyrosine content determined by Western blot analysis. The precision and limit of detection of the assay were also evaluated and were found to be approximately 10% (relative standard deviation) and 5 fmol on-column, respectively. These results demonstrate the utility of the NRP method for quantitative analyses of posttranslation modifications, in terms of broad applicability, ease of experimental design, sensitivity, and precision.

Quantitative dynamics of site-specific protein phosphorylation determined using liquid chromatography electrospray ionization mass spectrometry.

We have developed and validated a method that uses liquid chromatography/electrospray ionization-mass spectrometry to quantify site-specific protein phosphorylation. The method uses selected ion monitoring to determine the chromatographic peak areas of specific tryptic peptides from the protein of interest. The extent of phosphorylation is determined from the ratio of the phosphopeptide peak area to the peak area of an unmodified reference peptide that acts as internal standard, correcting for variations in protein amounts and peptide recovery in the digest preparation procedure. As a result, we refer to this protocol as the native reference peptide method. Mole of phosphate at the selected site per mole of protein is obtained from this ratio, using calibration curves of synthetic peptides to determine relative responses. Our method begins with protein separation by SDS-PAGE and is carried out on amounts of peptide produced by an in-gel digestion of single Coomassie blue-stained bands. To illustrate the utility of the method and provide validation, we used cardiac troponin I as analyte and monitored the time course of a protein kinase C betaII reaction. Those analyses appropriately demonstrate the time-dependent increase of phosphorylation at a PKC-preferred site, Ser44 in the peptide 41ISASPR45 and the concomitant consumption of the nonphosphorylated peptide. We believe that this method provides a novel tool to directly measure specific phosphorylation sites in proteins in different physiological states and expect that the method will be adaptable not only to a variety of samples types (i.e., culture cells, tissues, etc.) but to a variety of posttranslation modifications as well.