pFind Studio: a computational solution for mass spectrometry-based proteomics
2018
Journal of proteome research2018. Hu, A et al.
Box 355065,Foege Bldg,S220B,3720 15th Ave NE, Seattle, WA 98195 USA.
ABSTRACT:In data independent acquisition (DIA) mass spectrometry, precursor scans are interleaved with wide-window fragmentation scans, resulting in complex fragmentation spectra containing multiple coeluting peptide species. In this setting, detecting the isotope distribution profiles of intact peptides in the precursor scans can be a critical initial step in accurate peptide detection and quantification. This peak detection step is particularly challenging when the isotope peaks associated with two different peptide species overlap-or interfere-with one another. We propose a regression model, called Siren, to detect isotopic peaks in precursor DIA data that can explicitly account for interference. We validate Siren's peak-calling performance on a variety of data sets by counting how many of the peaks Siren identifies are associated with confidently detected peptides. In particular, we demonstrate that substituting the Siren regression model in place of the existing peak-calling step in DIA-Umpire leads to improved overall rates of peptide detection.
Use: pParse
Chemical Science2018. Park, DD et al.
Univ Calif Davis, Dept Chem, Davis, CA 95616 USA.
ABSTRACT:Given that unnatural sugar expression is metabolically achieved, the kinetics and disposition of incorporation can lend insight into the temporal and localization preferences of sialylation across the cell surface. However, common detection schemes lack the ability to detail the molecular diversity and distribution of target moieties. Here we employed a mass spectrometric approach to trace the placement of azido sialic acids on membrane glycoconjugates, which revealed substantial variations in incorporation efficiencies between N-/O-glycans, glycosites, and glycosphingolipids. To further explore the propensity for sialylation, we subsequently mapped the native glycome of model epithelial cell surfaces and illustrate that while glycosylation sites span broadly across the extracellular region, a higher number of heterogeneous glycoforms occur on sialylated sites closest to the transmembrane domain. Beyond imaging techniques, this integrative approach provides unprecedented details about the frequency and structure-specific distribution of cell surface sialylation, a critical feature that regulates cellular interactions and homeostatic pathways.
Use: pGlyco
Analyst2018. Xue, Y et al.
Fudan Univ, Dept Chem, Shanghai 200433, Peoples R China.
ABSTRACT:Glycosylation is a crucial process in protein biosynthesis. However, the analysis of glycopeptides through MS remains challenging due to the microheterogeneity and macroheterogeneity of the glycoprotein. Selective enrichment of glycopeptides from complex samples prior to MS analysis is essential for successful glycoproteome research. In this work, we systematically investigated the behaviors and performances of boronic acid chemistry, ZIC-HILIC, and PGC of glycopeptide enrichment to promote understanding of these methods. We also optimized boronic acid chemistry and ZIC-HILIC enrichment methods and applied them to enrich glycopeptides from mouse liver. The intact N-glycopeptides were interpreted using the in-house analysis software pGlyco 2.0. We found that boronic acid chemistry in this study preferred to capture glycopeptides with high mannose glycans, ZIC-HILIC enriched most N-glycopeptides and did not show significant preference during enrichment and PGC was not suitable for separating glycopeptides with a long amino acid sequence. We performed a detailed study on the behaviors and performances of boronic acid chemistry, ZIC-HILIC, and PGC enrichment methods and provide a better understanding of enrichment methods for further glycoproteomics research.
Use: pGlyco
Frontiers in Immunology2018. Su, YL et al.
Cent China Normal Univ, Sch Life Sci, Hubei Key Lab Genet Regulat & Integrat Biol, Wuhan, Hubei, Peoples R China.
ABSTRACT:Immunoglobulin M (IgM) is the major antibody in teleost fish and plays an important role in humoral adaptive immunity. The N-linked carbohydrates presenting on IgM have been well documented in higher vertebrates, but little is known regarding site-specific N-glycan characteristics in teleost IgM. In order to characterize these site-specific N-glycans, we conducted the first study of the N-glycans of each glycosylation site of the grass carp serum IgM. Among the four glycosylation sites, the Asn-262, Asn-303, and Asn-426 residues were efficiently glycosylated, while Asn-565 at the C-terminal tailpiece was incompletely occupied. A striking decrease in the level of occupancy at the Asn-565 glycosite was observed in dimeric IgM compared to that in monomeric IgM, and no glycan occupancy of Asn-565 was observed in tetrameric IgM. Glycopeptide analysis with liquid chromatography-electrospray ionization tandem mass spectrometry revealed mainly complex-type glycans with substantial heterogeneity, with neutral; monosialyl-, disialyl- and trisialylated; and fucosyl-and non-fucosyl-oligosaccharides conjugated to grass carp serum IgM. Glycan variation at a single site was greatest at the Asn-262 glycosite. Unlike IgMs in other species, only traces of complex-type and no high-mannose glycans were found at the Asn-565 glycosite. Matrix-assisted laser desorption ionization analysis of released glycans confirmed the overwhelming majority of carbohydrates were of the complex-type. These results indicate that grass carp serum IgM exhibits unique N-glycan features and highly processed oligosaccharides attached to individual glycosites.
Use: pGlyco
Talanta2018. Zhang, Y et al.
Natl Ctr Prot Sci Beijing, Beijing Inst Life, Beijing Proteome Res Ctr, State Key Lab Prote, Beijing 102206, Peoples R China.
ABSTRACT:It is challenging to capture N-glycopeptides with high recovery and high specificity from complicated biosystems. Herein, we present a facile and economical procedure to generate a novel self-assembling 4-Mercaptobenzene boronic acid functionalized and Au-doped Straticulate C3N4 (MASC), with enhanced affinity capability towards glycopeptides. The materials possess low pH value adaptation, high hydrophilicity and stability, good repeatability and recyclability, and provided high selectivity (1:100), low limit of detection (0.33 fmol/mu L), high enrichment efficiency (similar to 80%) and high recovery rate (similar to 90%) towards glycopeptides. The materials can capture glycopeptides unbiasedly, as demonstrated by the identification of 37 glycopeptides from IgG and 21 glycopeptides from horseradish peroxidase (HRP). The performance of MASC on human urine and serum glycoproteome analysis was also tested. An average of 1465 glycopeptides from 839 glycoproteins and 1553 glycopeptides from 884 glycoproteins were identified from female and male urine samples in a single mass spectrometry analysis. O-glycopeptides from human urine were also significantly enriched. Additionally, 463 glycopeptides assigned to 209 glycoproteins were identified from 5 pi, of human serum. All of these results indicate that MASC presents a good performance and applicability in the field of glycoproteomic research.
Use: pGlyco
Nature Protocol2018. Yuanhui Ma et al.
Departments of Molecular Medicine and Neurobiology, The Scripps Research Institute, La Jolla, CA, USA.
ABSTRACT:Measuring proteome response to perturbations is critical for understanding the underlying mechanisms involved. Traditional quantitative proteomic methods are limited by the large numbers of proteins in the proteome and the mass spectrometers dynamic range. A previous method uses the biorthogonal reagent azidohomoalanine (AHA), a methionine analog, for labeling, enrichment and detection of newly synthesized proteins (NSPs). Newly synthesized AHA proteins can be coupled to biotin via CuAAC-mediated click chemistry and enriched using avidin-based affinity purification. The combination of AHA-mediated NSP labeling with metabolic stable isotope labeling allows quantitation of low-abundant, newly secreted proteins by mass spectrometry (MS). However, the resulting multiplicity of labeling complicates NSP analysis. We developed a new NSP quantification strategy, called HILAQ (heavy isotopelabeled azidohomoalanine quantification), that uses a heavy isotopelabeled AHA molecule to enable NSP labeling, enrichment, identification and quantification. In addition, the AHA-peptide enrichment used in HILAQ improves both the identification and quantification of NSPs over AHA-protein enrichment. Here, we provide a description of the HILAQ method that includes procedures for (i) pulse-labeling and harvesting NSPs; (ii) addition of biotin by click reaction; (iii) protein precipitation; (iv) protein digestion; (v) enrichment of AHA-biotin peptides by NeutrAvidin beads and four-step elution; (vi) MS analysis; and (vii) data analysis for the identification and quantification of NSPs by ProLuCID and pQuant. We demonstrate our HILAQ approach by identifying NSPs from cell cultures, but we anticipate that it can be adapted for applications in animal models. The whole protocol takes ~6 d to complete.
Use: pQuant
Journal of molecular and cellular cardiology2018. McClatchy, DB et al.
Scripps Res Inst, Dept Mol Med, La Jolla, CA 92037 USA.
ABSTRACT:Cardiac remodeling (CR) is a complex dynamic process common to many heart diseases. CR is characterized as a temporal progression of global adaptive and maladaptive perturbations. The complex nature of this process clouds a comprehensive understanding of CR, but greater insight into the processes and mechanisms has potential to identify new therapeutic targets. To provide a deeper understanding of this important cardiac process, we applied a new proteomic technique, PALM (Pulse Azidohomoalanine in Mammals), to quantitate the newly synthesized protein (NSP) changes during the progression of isoproterenol (ISO)-induced CR in the mouse left ventricle. This analysis revealed a complex combination of adaptive and maladaptive alterations at acute and prolonged time points including the identification of proteins not previously associated with CR. We also combined the PALM dataset with our published protein turnover rate dataset to identify putative biochemical mechanisms underlying CR. The novel integration of analyzing NSPs together with their protein turnover rates demonstrated that alterations in specific biological pathways (e.g., inflammation and oxidative stress) are produced by differential regulation of protein synthesis and degradation.
Use: pQuant
Journal of molecular and cellular cardiology2018. McClatchy, DB et al.
Scripps Res Inst, Dept Mol Med, La Jolla, CA 92037 USA.
ABSTRACT:Cardiacremodeling(CR) is a complex dynamic process common to many heart diseases. CR is characterized as atemporalprogressionofglobal adaptive andmaladaptiveperturbations. The complex natureofthis process clouds a comprehensive understandingofCR, but greater insight into the processes and mechanisms has potential to identify new therapeutic targets. To provide a deeper understandingofthis importantcardiacprocess, we applied a new proteomic technique, PALM (Pulse AzidohomoalanineinMammals), to quantitate the newly synthesizedprotein(NSP) changes during the progressionofisoproterenol (ISO)-induced CRinthe mouse left ventricle. Thisanalysisrevealed a complex combinationofadaptive andmaladaptivealterations at acute and prolonged time points including the identificationofproteins not previously associated with CR. We also combined the PALM dataset with our publishedproteinturnover rate dataset to identify putative biochemical mechanisms underlying CR. The novel integrationofanalyzing NSPs together with theirproteinturnover rates demonstrated that alterationsinspecific biological pathways (e.g., inflammation and oxidative stress) are produced by differential regulationofproteinsynthesis and degradation.
Use: pQuant