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Label-free quantification is a powerful tool for the measurement of protein abundances by mass spectrometric methods. To maximize quantifiable identifications, MS(1)-based methods must balance the collection of survey scans and fragmentation spectra while maintaining reproducible extracted ion chromatograms (XIC). Here we present a method which increases the depth of proteome coverage over replicate data-dependent experiments without the requirement of additional instrument time or sample prefractionation. Sampling depth is increased by restricting precursor selection to a fraction of the full MS(1) mass range for each replicate; collectively, the m/z segments of all replicates encompass the full MS(1) range. Although selection windows are narrowed, full MS(1) spectra are obtained throughout the method, enabling the collection of full mass range MS(1) chromatograms such that label-free quantitation can be performed for any peptide in any experiment. We term this approach "binning" or "tiling" depending on the type of m/z window utilized. By combining the data obtained from each segment, we find that this approach increases the number of quantifiable yeast peptides and proteins by 31% and 52%, respectively, when compared to normal data-dependent experiments performed in replicate.

Plant cell wall pectic polysaccharides are arguably the most complex carbohydrates in nature. Progress in understanding pectin synthesis has been slow due to its complex structure and difficulties in purifying and expressing the low-abundance, Golgi membrane-bound pectin biosynthetic enzymes. Arabidopsis galacturonosyltransferase (GAUT) 1 is an α-1,4-galacturonosyltransferase (GalAT) that synthesizes homogalacturonan (HG), the most abundant pectic polysaccharide. We now show that GAUT1 functions in a protein complex with the homologous GAUT7. Surprisingly, although both GAUT1 and GAUT7 are type II membrane proteins with single N-terminal transmembrane-spanning domains, the N-terminal region of GAUT1, including the transmembrane domain, is cleaved in vivo. This raises the question of how the processed GAUT1 is retained in the Golgi, the site of HG biosynthesis. We show that the anchoring of GAUT1 in the Golgi requires association with GAUT7 to form the GAUT1:GAUT7 complex. Proteomics analyses also identified 12 additional proteins that immunoprecipitate with the GAUT1:GAUT7 complex. This study provides conclusive evidence that the GAUT1:GAUT7 complex is the catalytic core of an HG:GalAT complex and that cell wall matrix polysaccharide biosynthesis occurs via protein complexes. The processing of GAUT1 to remove its N-terminal transmembrane domain and its anchoring in the Golgi by association with GAUT7 provides an example of how specific catalytic domains of plant cell wall biosynthetic glycosyltransferases could be assembled into protein complexes to enable the synthesis of the complex and developmentally and environmentally plastic plant cell wall.

With the recent completion of the Brugia malayi genome, proteomics offers a new resource for a deeper understanding of the biology of filarial parasites. We employed 2-dimensional (2D) gel electrophoresis followed by peptide mass fingerprinting on a matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) mass spectrometer to identify Brugia adult worm proteins and then determined which proteins were recognized by the host humoral immune response. We identified 18 unique proteins, several of which were determined to be antigenic by immunoblot. The proteins identified here may contribute to future studies to analyze the transmission and pathogenesis of lymphatic filariasis.

Robust quantification is an essential component of comparative -omic strategies. In this regard, glycomics lags behind proteomics. Although various isotope-tagging and direct quantification methods have recently enhanced comparative glycan analysis, a cell culture labeling strategy, that could provide for glycomics the advantages that SILAC provides for proteomics, has not been described. Here, we report the development of IDAWG, Isotopic Detection of Aminosugars With Glutamine, for the incorporation of differential mass tags into the glycans of cultured cells. In this method, culture media containing amide-(15)N-Gln is used to metabolically label cellular aminosugars with heavy nitrogen. Because the amide side chain of Gln is the sole source of nitrogen for the biosynthesis of GlcNAc, GalNAc, and sialic acid, we demonstrate that culturing mouse embryonic stems cells for 72 h in the presence of amide-(15)N-Gln media results in nearly complete incorporation of (15)N into N-linked and O-linked glycans. The isotopically heavy monosaccharide residues provide additional information for interpreting glycan fragmentation and also allow quantification in both full MS and MS/MS modes. Thus, IDAWG is a simple to implement, yet powerful quantitative tool for the glycomics toolbox.

Although brain tumors are classified as if their lineage were well understood, the relationship between the molecular events that specify neural cell lineage and brain tumors remains enigmatic. Traditionally, cell surface membrane antigens have served as biomarkers that distinguish brain tumor origin and malignancy. In this study, membrane proteins were identified from a terminally differentiated mouse astrocyte (AC) and CT-2A astrocytoma (CT-2A) cell line using liquid-chromatography coupled with tandem mass spectrometry (LC-MS/MS). A total of 321 and 297 protein groups with at least one unique peptide were identified in the AC and CT-2A cells. Using a label-free quantitative MS approach, 25 plasma membrane proteins in CT-2A were found significantly up- or down-regulated compared with those in AC. Three of the up-regulated proteins, chondroitin sulfate proteoglycan-4 (Cspg4), interferon-induced transmembrane protein-2 (IFITM2) and -3 (IFITM3) were further validated by semi-quantitative RT-PCR analysis. In addition, a third member of the IFITM family, interferon-induced transmembrane protein-1 (IFITM1) was also analyzed. Expression of Cspg4, IFITM1 and IFITM3 was significantly greater in the CT-2A cells than that in the AC cells. Interestingly, Cspg4, also known as neuronal/glial 2 (NG2) proteoglycan in human, is an oligodendrocyte progenitor marker. Therefore, our data suggest that the CT-2A tumor may be derived from NG2 glia rather than from fully differentiated astrocytes. Moreover, the CT-2A cells also express a series of interferon-induced signature proteins that may be specific to this tumor. These data highlight the utility of LC-MS/MS for the identification of brain tumor membrane biomarkers.

The study of glycosylation patterns (glycomics) in biological samples is an emerging field that can provide key insights into cell development and pathology. A current challenge in the field of glycomics is to determine how to quantify changes in glycan expression between different cells, tissues, or biological fluids. Here we describe a novel strategy, quantitation by isobaric labeling (QUIBL), to facilitate comparative glycomics. Permethylation of a glycan with (13)CH 3I or (12)CH 2DI generates a pair of isobaric derivatives, which have the same nominal mass. However, each methylation site introduces a mass difference of 0.002922 Da. As glycans have multiple methylation sites, the total mass difference for the isobaric pair allows separation and quantitation at a resolution of approximately 30000 m/Delta m. N-Linked oligosaccharides from a standard glycoprotein and human serum were used to demonstrate that QUIBL facilitates relative quantitation over a linear dynamic range of 2 orders of magnitude and permits the relative quantitation of isomeric glycans. We applied QUIBL to quantitate glycomic changes associated with the differentiation of murine embryonic stem cells to embryoid bodies.

The use of Fourier transform mass spectrometry (FTMS) to monitor noncovalent complex formation in the gas phase under native conditions between the Link module from human tumor necrosis factor stimulated gene-6 (Link_TSG6) and hyaluronan (HA) oligosaccharides is reported. In particular, a titration experiment with increasing concentrations of octasaccharide (HA(8)) to protein produced a noncovalent complex with 1:1 stoichiometry when the oligosaccharide was in molar excess. However, in the presence of a molar excess of tetrasaccharide (HA(4)) nearly all proteins and oligosaccharides were observed in their unbound charge states. These results are consistent with solution-phase properties for this interaction in which HA(8), but not HA(4), supports high affinity Link_TSG6 binding. Hydrogen/deuterium amide exchange mass spectrometry (H/D-EX MS) was also utilized to investigate the level of global deuterium incorporation, over time, for Link_TSG6 in both the absence and presence of HA(8). After dilution into quenching conditions, deuterium incorporation reached limiting asymptotic values of 37 and 26 deuterons for the free and bound protein at 240 and 480 min, respectively, indicating that the oligosaccharide interferes with amide exchange on binding. To detect sequence-specific deuterium incorporation, pepsin digestion of Link_TSG6 in both the absence and presence of HA(8) was performed. A level of deuterium incorporation of 10-30% was observed for peptides analyzed in free Link_TSG6. Interestingly, HA(8) blocked some sites of proteolysis in Link_TSG6 compared to the free protein. Molecular modeling indicated that amino acids proximal to the ligand correlated with regions of the protein that were resistant to enzymatic digestion. Of the peptides that could be analyzed by H/D-EX MS in the presence of the ligand, a 30-60% reduction in deuterium incorporation, relative to the free protein, was observed, even for those sequences not directly involved in HA binding. These results support the utility of FTMS as a method for the characterization of protein-carbohydrate interactions., (Copyright (c) 2006 John Wiley & Sons, Ltd.)

Herein we detail the first glycoproteomic analysis of a human pathogen. We describe an approach that enables the identification of organelle and cell surface N-linked glycoproteins from Trypanosoma cruzi, the causative agent of Chagas' disease. This approach is based on a subcellular fractionation protocol to produce fractions enriched in either organelle or plasma membrane/cytoplasmic proteins. Through lectin affinity capture of the glycopeptides from each subcellular fraction and stable isotope labeling of the glycan attachment sites with H(2)18O, we unambiguously identified 36 glycosylation sites on 35 glycopeptides which mapped to 29 glycoproteins. We also present the first expression evidence for 11 T. cruzi specific glycoproteins and provide experimental data indicating that the mucin associated surface protein family (MASP) and dispersed gene family (DGF-1) are post-translationally modified by N-linked glycans.

To complement the sequencing of the three kinetoplastid genomes reported in this issue, we have undertaken a whole-organism, proteomic analysis of the four life-cycle stages of Trypanosoma cruzi. Peptides mapping to 2784 proteins in 1168 protein groups from the annotated T. cruzi genome were identified across the four life-cycle stages. Protein products were identified from >1000 genes annotated as "hypothetical" in the sequenced genome, including members of a newly defined gene family annotated as mucin-associated surface proteins. The four parasite stages appear to use distinct energy sources, including histidine for stages present in the insect vectors and fatty acids by intracellular amastigotes.

MS/MS and database searching has emerged as a valuable technology for rapidly analyzing protein expression, localization, and post-translational modifications. The probability-based search engine Mascot has found widespread use as a tool to correlate tandem mass spectra with peptides in a sequence database. Although the Mascot scoring algorithm provides a probability-based model for peptide identification, the independent peptide scores do not correlate with the significance of the proteins to which they match. Herein, we describe a heuristic method for organizing proteins identified at a specified false-discovery rate using Mascot-matched peptides. We call this method PROVALT, and it uses peptide matches from a random database to calculate false-discovery rates for protein identifications and reduces a complex list of peptide matches to a nonredundant list of homologous protein groups. This method was evaluated using Mascot-identified peptides from a Trypanosoma cruzi epimastigote whole-cell lysate, which was separated by multidimensional LC and analyzed by MS/MS. PROVALT was then compared with the two traditional methods of protein identification when using Mascot, the single peptide score and cumulative protein score methods, and was shown to be superior to both in regards to the number of proteins identified and the inclusion of lower scoring nonrandom peptide matches.

We describe an algorithm which modifies a protein database such that during a database search deamidation is limited to asparagines strictly contained within the N-glycosylation consensus sequence. The modified database was evaluated using a dataset created from the shotgun proteomic analysis of N-linked glycopeptides from human blood serum. We demonstrate that the application of the modified database eliminates incorrect glycopeptide assignments, reduces the peptide false-discovery rate, and eliminates the need for manual validation of glycopeptide identifications., ((c) 2005 John Wiley & Sons, Ltd.)

The fungus Botrytis cinerea is a ubiquitous plant pathogen that infects more than 200 different plant species and causes substantial economic losses in a wide range of agricultural crops and harvested products. Endopolygalacturonases (EPGs) are among the first array of cell-wall-degrading enzymes secreted by fungi during infection. Up to 13 EPG glycoforms have been described for B. cinerea. The presence of multiple N-linked glycosylation modifications in BcPG1-6 is predicted by their deduced amino acid sequences. In this work, the glycosylation sites and the attached oligosaccharide structures on BcPG6 were analyzed. The molecular mass of the intact glycoprotein was determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometric (MALDI-TOFMS) analysis. BcPG6 contains seven potential N-linked glycosylation sites. Occupancy of these glycosylation sites and the attached carbohydrate structures were analyzed by tryptic digestion followed by liquid chromatography/mass spectrometry (LC/MS) using a stepped orifice voltage approach. Five out of seven potential N-linked sites present in BcPG6 were determined to be occupied by high-mannose-type oligosaccharides. Four of them were readily determined to be at Asn58 (T3 peptide), Asn198 (T7 peptide), Asn237 (T9 peptide) and Asn256 (T11 peptide), respectively. Another was located on the T8 peptide, which contained two potential N-linked sites, Asn224 and Asn227 (SNNN224VTN227ITFK). LC/MS/MS of a sample treated with N-glycanase placed the glycan in this peptide at Asn224 rather than at Asn227. The potential glycosylation site on Asn146 (T6 peptide) was not glycosylated. In addition, two disulfide bonds were observed, linking the Cys residues within the T13 and T16 peptides., (Copyright (c) 2005 John Wiley & Sons, Ltd.)