pFind Studio: a computational solution for mass spectrometry-based proteomics
2020
Genomics, proteomics & bioinformatics2020. Lin, XH et al.
Chinese Acad Sci, Inst Hydrobiol, State Key Lab Freshwater Ecol & Biotechnol, Wuhan 430072, Peoples R China.
ABSTRACT:Protein lysine methylation is a prevalent post-translational modification (PTM) and plays critical roles in all domains of life. However, its extent and function in photosynthetic organisms are still largely unknown. Cyanobacteria are a large group of prokaryotes that carry out oxygenic photosynthesis and are applied extensively in studies of photosynthetic mechanisms and environmental adaptation. Here we integrated propionylation of monomethylated proteins, enrichment of the modified peptides, and mass spectrometry (MS) analysis to identify monomethylated proteins in Synechocystis sp. PCC 6803 (Synechocystis). Overall, we identified 376 monomethylation sites in 270 proteins, with numerous monomethylated proteins participating in photosynthesis and carbon metabolism. We subsequently demonstrated that CpcM, a previously identified asparagine methyl-transferase in Synechocystis, could catalyze lysine monomethylation of the potential aspartate amino-transferase 5110480 both in vivo and in vitro and regulate the enzyme activity of 5110480. The loss of CpcM led to decreases in the maximum quantum yield in primary photosystem II (PSII) and the efficiency of energy transfer during the photosynthetic reaction in Synechocystis. We report the first lysine monomethylome in a photosynthetic organism and present a critical database for functional analyses of monomethylation in cyanobacteria. The large number of monomethylated proteins and the identification of CpcM as the lysine methyltransferase in cyanobacteria suggest that reversible methylation may influence the metabolic process and photosynthesis in both cyanobacteria and plants.
Use: pFind
Genomics, proteomics & bioinformatics2020. Lin, XH et al.
Chinese Acad Sci, Inst Hydrobiol, State Key Lab Freshwater Ecol & Biotechnol, Wuhan 430072, Peoples R China.
ABSTRACT:Protein lysine methylation is a prevalent post-translational modification (PTM) and plays critical roles in all domains of life. However, its extent and function in photosynthetic organisms are still largely unknown. Cyanobacteria are a large group of prokaryotes that carry out oxygenic photosynthesis and are applied extensively in studies of photosynthetic mechanisms and environmental adaptation. Here we integrated propionylation of monomethylated proteins, enrichment of the modified peptides, and mass spectrometry (MS) analysis to identify monomethylated proteins in Synechocystis sp. PCC 6803 (Synechocystis). Overall, we identified 376 monomethylation sites in 270 proteins, with numerous monomethylated proteins participating in photosynthesis and carbon metabolism. We subsequently demonstrated that CpcM, a previously identified asparagine methyl-transferase in Synechocystis, could catalyze lysine monomethylation of the potential aspartate amino-transferase 5110480 both in vivo and in vitro and regulate the enzyme activity of 5110480. The loss of CpcM led to decreases in the maximum quantum yield in primary photosystem II (PSII) and the efficiency of energy transfer during the photosynthetic reaction in Synechocystis. We report the first lysine monomethylome in a photosynthetic organism and present a critical database for functional analyses of monomethylation in cyanobacteria. The large number of monomethylated proteins and the identification of CpcM as the lysine methyltransferase in cyanobacteria suggest that reversible methylation may influence the metabolic process and photosynthesis in both cyanobacteria and plants.
Use: pFind
Current bioinformatics2020. Rolfs, Z et al.
Univ Wisconsin, Dept Chem, 1101 Univ Ave, Madison, WI 53706 USA.
ABSTRACT:Background: The identification of non-specifically cleaved peptides in proteomics and peptidomics poses a significant computational challenge. Current strategies for the identification of such peptides are typically time-consuming and hinder routine data analysis. Objective: We aimed to design an algorithm that would improve the speed of semi- and nonspecific enzyme searches and could be applied to existing search programs. Methods: We developed a novel search algorithm that leverages fragment-ion redundancy to simultaneously search multiple non-specifically cleaved peptides at once. Briefly, a theoretical peptide tandem mass spectrum is generated using only the fragment-ion series from a single terminus. This spectrum serves as a proxy for several shorter theoretical peptides sharing the same terminus. After database searching, amino acids are removed from the opposing terminus until the observed and theoretical precursor masses match within a given mass tolerance. Results: The algorithm was implemented in the search program MetaMorpheus and found to perform an order of magnitude faster than the traditional MetaMorpheus search and produce superior results. Conclusion: We report a speedy non-specific enzyme search algorithm that is open-source and enables search programs to utilize fragmention redundancy to achieve a notable increase in search speed.
Use: pFind
STAR Protocols2020. Li, Yi et al.
Peking University
ABSTRACT:Alk-Ph is a clickable APEX2 substrate developed for spatially restricted protein/RNA labeling in intact yeast cells. Alk-Ph is more water soluble and cell wall permeable than biotin-phenol substrate, allowing more efficient profiling of the subcellular proteome in microorganisms. We describe the protocol for Alk-Ph probe synthesis, APEX2 expression, and protein/RNA labeling in yeast and the workflow for quantitative proteomic experiments and data analysis. Using the yeast mitochondria as an example, we provide guidelines to achieve high-resolution mapping of subcellular yeast proteome and transcriptome. For complete details on the use and execution of this protocol, please refer to Li etal. (2020). 2020 The Author(s).
Use: pQuant; pFind
STAR Protocols2020. Li, Yi et al.
Peking University
ABSTRACT:Alk-Ph is a clickable APEX2 substrate developed for spatially restricted protein/RNA labeling in intact yeast cells. Alk-Ph is more water soluble and cell wall permeable than biotin-phenol substrate, allowing more efficient profiling of the subcellular proteome in microorganisms. We describe the protocol for Alk-Ph probe synthesis, APEX2 expression, and protein/RNA labeling in yeast and the workflow for quantitative proteomic experiments and data analysis. Using the yeast mitochondria as an example, we provide guidelines to achieve high-resolution mapping of subcellular yeast proteome and transcriptome. For complete details on the use and execution of this protocol, please refer to Li etal. (2020). 2020 The Author(s).
Use: pQuant; pFind
Genomics, proteomics & bioinformatics2020. Zhu, Tiansheng et al.
Westlake Univ, Zhejiang Prov Lab Life Sci & Biomed, Hangzhou 310024, Peoples R China; Westlake Univ, Sch Life Sci, Key Lab Struct Biol Zhejiang Prov, Hangzhou 310024, Peoples R China; Westlake Inst Adv Study, Inst Basic Med Sci, Hangzhou 310024, Peoples R China
ABSTRACT:To address the increasing need for detecting and validating protein biomarkers in clinical specimens, mass spectrometry (MS)-based targeted proteomic techniques, including the selected reaction monitoring (SRM), parallel reaction monitoring (PRM), and massively parallel dataindependent acquisition (DIA), have been developed. For optimal performance, they require the fragment ion spectra of targeted peptides as prior knowledge. In this report, we describe a MS pipeline and spectral resource to support targeted proteomics studies for human tissue samples. To build the spectral resource, we integrated common open-source MS computational tools to assemble a freely accessible computational workflow based on Docker. We then applied the workflow to generate DPHL, a comprehensive DIA pan-human library, from 1096 data-dependent acquisition (DDA) MS raw files for 16 types of cancer samples. This extensive spectral resource was then applied to a proteomic study of 17 prostate cancer (PCa) patients. Thereafter, PRM validation was applied to a larger study of 57 PCa patients and the differential expression of three proteins in prostate tumor was validated. As a second application, the DPHL spectral resource was applied to a study consisting of plasma samples from 19 diffuse large B cell lymphoma (DLBCL) patients and 18 healthy control subjects. Differentially expressed proteins between DLBCL patients and healthy control subjects were detected by DIA-MS and confirmed by PRM. These data demonstrate that the DPHL supports DIA and PRM MS pipelines for robust protein biomarker discovery. DPHL is freely accessible at https://www.iprox.org/page/project.html?id=IPX0001400000.
Use: pFind
Journal of proteome research2020. Gao, Huanhuan et al.
Westlake Inst Adv Study, Inst Basic Med Sci, Hangzhou 310024, Zhejiang, Peoples R China; Westlake Univ, Sch Life Sci, Key Lab Struct Biol Zhejiang Prov, Hangzhou 310024, Zhejiang, Peoples R China
ABSTRACT:Pressure cycling technology (PCT)-assisted tissue lysis and digestion have facilitated reproducible and high-throughput proteomic studies of both fresh-frozen (FF) and formalin-fixed paraffin-embedded (FFPE) tissue of biopsy scale for biomarker discovery. Here, we present an improved PCT method accelerating the conventional procedures by about two-fold without sacrificing peptide yield, digestion efficiency, peptide, and protein identification. The time required for processing 16 tissue samples from tissues to peptides is reduced from about 6 to about 3 h. We analyzed peptides prepared from FFPE hepatocellular carcinoma (HCC) tissue samples by the accelerated PCT method using multiple MS acquisition methods, including short-gradient SWATH-MS, PulseDIA-MS, and 10-plex TMT-based shotgun MS. The data showed that up to 8541 protein groups could be reliably quantified from the thus prepared peptide samples. We applied the accelerated sample preparation method to 25 pairs (tumorous and matched benign) of HCC samples followed by a single-shot, 15 min gradient SWATH-MS analysis. An average of 18 453 peptides from 2822 proteins were quantified in at least 20% samples in this cohort, while 1817 proteins were quantified in at least 50% samples. The data not only identified the previously known dysregulated proteins such as MCM7, MAPRE1, and SSRP1 but also discovered promising novel protein markers, including DRAP1 and PRMT5. In summary, we present an accelerated PCT protocol that effectively doubles the throughput of PCT-assisted sample preparation of biopsy-level FF and FFPE samples without compromising protein digestion efficiency, peptide yield, and protein identification.
Use: pFind
Molecular & cellular proteomics : MCP2020. Shu, Qingbo et al.
Laboratory of Protein and Peptide Pharmaceuticals & Proteomics Laboratory, Institute of Biophysics, Chinese Academy of Sciences
ABSTRACT:Large-scale identification of N-linked intact glycopeptides by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) in human serum is challenging because of the wide dynamic range of serum protein abundances, the lack of a complete serum N-glycan database and the existence of proteoforms. In this regard, a spectral library search method was presented for the identification of N-linked intact glycopeptides from N-linked glycoproteins in human serum with target-decoy and motif-specific false discovery rate (FDR) control. Serum proteins were firstly separated into low-abundance and high-abundance proteins by acetonitrile (ACN) precipitation. After digestion, the N-linked intact glycopeptides were enriched by hydrophilic interaction liquid chromatography (HILIC) and a portion of the enriched N-linked intact glycopeptides were processed by Peptide-N-Glycosidase F (PNGase F) to generate N-linked deglycopeptides. Both N-linked intact glycopeptides and deglycopeptides were analyzed by LC-MS/MS. From N-linked deglycopeptides data sets, 764 N-linked glycoproteins, 1699 N-linked glycosites and 3328 unique N-linked deglycopeptides were identified. Four types of N-linked glycosylation motifs (NXS/T/C/V, XP) were used to recognize the N-linked deglycopeptides. The spectra of these N-linked deglycopeptides were utilized for N-linked deglycopeptides library construction and identification of N-linked intact glycopeptides. A database containing 739 N-glycan masses was constructed and utilized during spectral library search for the identification of N-linked intact glycopeptides. In total, 526 N-linked glycoproteins, 1036 N-linked glycosites, 22,677 N-linked intact glycopeptides and 738 N-glycan masses were identified under 1% FDR, representing the most in-depth serum N-glycoproteome identified by LC-MS/MS at N-linked intact glycopeptide level. Copyright 2020 2020 Shu et al. Published by Elsevier Inc. All rights reserved.
Use: pParse; pGlyco; pFind
Scientific Reports2020. Liu, Yongtao et al.
Beijing Normal Univ, Dept Biochem & Mol Biol, Beijing Key Lab Gene Engn Drug & Biotechnol, Beijing, Peoples R China
ABSTRACT:In this study, two groups of human plasma proteome at different age groups (old and young) were used to perform a comparison of global chemical modifications, as determined by tandem mass spectrometry (MS/MS) combined with non-limiting modification identification algorithms. The sulfhydryl in the cysteine A total of 4 molecular modifications were found to have significant differences passing random grouping tests: the succinylation and phosphorylation modification of cysteine (Cys, C) and the modification of lysine (Lys, K) with threonine (Thr, T) were significantly higher in the old group than in the young group, while the carbamylation of lysine was lower in the young group. We speculate that there is an increase in certain modified proteins in the blood of the old people which, in turn, changes the function of those proteins. This change may be one of the reasons why old people are more likely than young people to be at risk for age-related diseases, such as metabolic diseases, cerebral and cardiovascular diseases, and cancer.
Use: pFind
Plant Physiology and Biochemistry2020. Li, Ling et al.
Chinese Acad Sci, Inst Bot, Key Lab Plant Resources, Beijing Bot Garden, Beijing 100093, Peoples R China; Chinese Acad Sci, Qingdao Inst, Shanghai Inst Technol Phys, Binhai Rd 50, Qingdao 264000, Peoples R China
ABSTRACT:In photosynthesis, the antenna system captures solar energy and transfers the excitations to photosystem II (PSII) core complex where charge separation, water splitting and oxygen evolution occur. In the evolution of photosynthesis from aquatic to terrestrial environments, the structure of PSII core complex was highly conserved while a variety of antenna forms became differentiated. In order to study the principles for energy transport from antenna to the PSII reaction center, we have explored whether the major light harvesting complex of PSII (LHCII) of higher plants can transfer energy to the cyanobacteria PSII core complexes (CC). For this purpose, LHCII from pea and CC from Thermosynechococcus vulcanus were isolated and co-reconstituted into liposome at LHCII:CC molar ratios of 2:1, 4:1 and 6:1, respectively. Chemical-cross linking followed by LC-MS/MS analysis confirmed the biochemical interaction between LHCII and CC in the liposome membrane. The analyses of 77 K fluorescence emission spectra and antenna cross section of PSII indicated that LHCII can transfer energy directly to the cyanobacterial CC. The study has laid the basis for further research on the mechanism of energy transfer from LHCII to PSII CC. This result may also open a new possibility for design and development of new artificial PSII in the application of solar energy conversion.
Use: pFind