Methodologies to enrich heterogeneous types of phosphopeptides are critical for comprehensive mapping the under-explored phosphoproteome. Taking advantage distinct binding affinities Ga3+ and Fe3+ phosphopeptides, we designed a metal-directed immobilized metal ion affinity chromatography sequential enrichment phosphopeptides. In Raji B cells, Ga3+-Fe3+-immobilized (IMAC) strategy displayed 1.5–3.5-fold superior phosphoproteomic coverage compared single IMAC (Fe3+, Ti4+, Ga3+, Al3+)....

Abstract Phosphoproteomics can provide insights into cellular signaling dynamics. To achieve deep and robust quantitative phosphoproteomics profiling for minute amounts of sample, we here develop a global strategy based on data-independent acquisition (DIA) mass spectrometry hybrid spectral libraries derived from data-dependent (DDA) DIA data. Benchmarking the method using 166 synthetic phosphopeptides shows high sensitivity (<0.1 ng), accurate site localization reproducible...

N-linked glycosylation is one of the predominant post-translational modifications involved in a number biological functions. Since experimental characterization glycosites challenging, glycosite prediction crucial. Several predictors have been made available and report high performance. Most them evaluate their performance at every asparagine protein sequences, not confined to N-X-S/T sequon. In this paper, we present N-GlyDE, two-stage tool trained on rigorously-constructed non-redundant...

Membrane proteins are crucial targets for cancer biomarker discovery and drug development. However, in addition to the inherent challenges of hydrophobicity low abundance, complete membrane proteome coverage clinical specimen is usually hindered by requirement large amount starting materials. Toward comprehensive proteomic profiling small amounts samples (10 μg), we developed high-pH reverse phase (Hp-RP) combined with stop-and-go extraction tip (StageTip) technique, as a fast (∼15 min.),...

Abstract Although EGFR tyrosine kinase inhibitors (TKIs) have demonstrated good efficacy in non-small-cell lung cancer (NSCLC) patients harboring mutations, most develop intrinsic and acquired resistance. We quantitatively profiled the phosphoproteome proteome of drug-sensitive drug-resistant NSCLC cells under gefitinib treatment. The construction a dose-dependent responsive kinase-substrate network 1548 phosphoproteins 3834 proteins revealed CK2-centric modules as dominant core for...

Despite significant efforts in the past decade toward complete mapping of human proteome, 3564 proteins (neXtProt, 09-2014) are still "missing proteins". Over one-third these missing annotated as membrane proteins, owing to their relatively challenging accessibility with standard shotgun proteomics. Using nonsmall cell lung cancer (NSCLC) a model study, we aim mine from disease-associated which may be largely under-represented. To increase identification coverage, employed Hp-RP StageTip...

Identifying peptides and proteins from mass spectrometry (MS) data, spectral library searching has emerged as a complementary approach to the conventional database searching. However, for spectrum-centric analysis of data-independent acquisition (DIA) not been widely exploited because existing search tools are mainly designed optimized data-dependent (DDA) data. We present Calibr, tool DIA data analysis. Calibr optimizes spectrum preprocessing pseudo MS2 spectra, generating an 8.11% increase...

Human embryonic stem cells (hESCs) have the capacity for self-renewal and multilineage differentiation, which are of clinical importance regeneration medicine. Despite significant progress hESC study, complete proteome atlas, especially surface protein composition, awaits delineation. According to latest release neXtProt database (January 17, 2018; 19 658 PE1, 2, 3, 4 human proteins), membrane proteins present major category (1047; 48%) among all 2186 missing (MPs). We conducted a deep...

MAGIC-web is the first web server, to best of our knowledge, that performs both untargeted and targeted analyses mass spectrometry-based glycoproteomics data for site-specific N-linked glycoprotein identification. The two modules, MAGIC MAGIC+, are designed analysis, respectively. implemented with previously proposed novel Y1-ion pattern matching method, which adequately detects Y1- Y0-ion without prior information proteins glycans, then generates in silico MS(2) spectra serve as input a...

Chromosome 4 is the fourth largest chromosome, containing approximately 191 megabases (∼6.4% of human genome) with 757 protein-coding genes. A number marker genes for many diseases have been found in this including genetic (e.g., hepatocellular carcinoma) and biomedical research (cardiac system, aging, metabolic disorders, immune cancer stem cell) related oncogenes, growth factors). As a pilot study chromosome 4-centric proteome project (Chr 4-HPP), we present here systematic analysis...

To confirm the existence of missing proteins, we need to identify at least two unique peptides with length 9–40 amino acids a protein in bottom-up mass-spectrometry-based proteomic experiments. However, an identified peptide protein, even high level confidence, could possibly coincide commonly observed due isobaric substitutions, mass modifications, alternative splice isoforms, or single acid variants (SAAVs). Besides variant (SAAV-containing peptides) also alternatively map other proteins...

Protein and peptide identification quantitation are essential tasks in proteomics research involve a series of steps analyzing mass spectrometry data. Trans-Proteomic Pipeline (TPP) provides wide range useful tools through its web interfaces for analyses such as sequence database search, statistical validation, quantitation. To utilize the powerful functionality TPP without need manual intervention to launch each step, we developed software tool, called WinProphet, create automatically...

Identifying single-amino-acid variants (SAVs) from mass spectrometry-based experiments is critical for validating single-nucleotide (SNVs) at the protein level to facilitate biomedical research. Currently, two approaches are usually applied convert SNV annotations into SAV-harboring sequences. One approach generates one sequence containing exactly SAV, and other all SAVs. However, they may neglect possibility of SAV combinations, e.g., haplotypes, existing in bio-samples. Therefore, it...

In proteogenomic studies, many genome-annotated events, for example, single amino acid variation (SAAV) and short INDEL, are often unobserved in shotgun proteomics. Therefore, we propose an analysis pipeline called LeTE-fusion (Le, peptide length; T, theoretical values; E, experimental data) to first investigate whether peptides with certain lengths observed more mass spectrometry (MS)-based proteomics, which may hinder identification causing difficulty detecting events. By applying on...

Lung adenocarcinoma (LUAD) patients in East Asia predominantly harbor oncogenic EGFR mutations. However, there remains a limited understanding of the biological characteristics and therapeutic vulnerabilities concurrent mutations other genes LUAD. Here, we performed comprehensive bioinformatics analyses on 88 treatment-naïve Asian LUAD patients. Based somatic mutation clustering, identified three subtypes: + TP53 co-mutation, mutation, multiple-gene mutation. A proteogenomic analysis among...

When conducting proteomics experiments to detect missing proteins and protein isoforms in the human proteome, it is desirable use a protease that can yield more unique peptides with properties amenable for mass spectrometry analysis. Though trypsin currently most widely used protease, some only limited number of by digestion. Other proteases multiple have been applied reported studies increase identified sequence coverage. To facilitate selection proteases, we developed web-based resource,...

Mass spectrometry-based proteomics using isobaric labeling for multiplex quantitation has become a popular approach proteomic studies. We present Multi-Q 2, an isobaric-labeling tool which can yield the largest coverage and improved accuracy compared to three state-of-the-art methods. 2 supports identification results from several data analysis platforms quantitation, offering up 12% improvement in accepting multiple search engines when with MaxQuant PatternLab. It is equipped various...

Concatenated target-decoy database searches are commonly used in proteogenomic research for variant peptide identification. Currently, protein-based and peptide-based sequence databases applied to store sequences searches. The records a full-length wild-type protein but using the given events replace original amino acids, whereas retains only silico digested peptides containing variants. However, performance of applying various decoy generation methods on is still unclear, compared database....

Adopting proteogenomics approach to validate single nucleotide variation events by identifying corresponding amino acid variant peptides from mass spectrometry (MS)-based proteomics data facilitates translational and clinical research. Although are usually identified MS with a stringent false discovery rate (FDR), FDR control could fail eliminate dubious results caused several issues; thus, postexamination is required. However, comprehensive postexaminations of identification still lacking....