pFind Studio: a computational solution for mass spectrometry-based proteomics
2013 or earlier
Electrophoresis2013. Ma, C et al.
Beijing Inst Technol, Sch Life Sci & Technol, Beijing 100081, Peoples R China.
ABSTRACT:N-linked glycosylation is an important protein posttranslational modification that is involved in numerous biological processes. Different methods, including chemical reaction and affinity interaction, have been developed to enrich glycosylated peptides or proteins from biological systems. However, due to the common occurrence of low glycosites occupancy in proteins and the low efficiency of enrichment approaches, only a small fraction of protein glycosites have been reported. In this study, we combined the glycopeptide enrichment strategy for broad analysis of human serum N-glycoproteins using a tandem enrichment method coupling lectin affinity capture with HILIC. This strategy was applied to profile the human serum N-linked glycoproteome, and it resulted in 32 and 14% more N-glycosites than could be identified with the common lectin affinity capture or HILIC approaches, respectively. With an additional dimension of glycopeptides separation using high-pH reversed phase liquid chromatography or off-gel electrophoresis, the number of identified glycosites was increased by 3.1-fold and 1.8-fold, respectively. These results demonstrate that tandem enrichment methods, especially when followed by high-pH reversed-phase prefractionation, can greatly improve the power of N-glycoproteome analysis. In total, 615 N-glycosites from 312 glycoproteins (protein group) were mapped using high-accuracy mass spectrometry.
Use: pFind
Journal of proteome research2013. Yang, MK et al.
Chinese Acad Sci, Inst Hydrobiol, Key Lab Algal Biol, Wuhan 430072, Peoples R China.
ABSTRACT:Increasing evidence shows that protein phosphorylation on serine (Ser), threonine (Thr), and tyrosine (Tyr) residues is one of the major post-translational modifications in the bacteria, involved in regulating a myriad of physiological processes. Cyanobacteria are one of the largest groups of bacteria and are the only prokaryotes capable of oxygenic photosynthesis. Many cyanobacteria strains contain unusually high numbers of protein kinases and phosphatases with specificity on Ser, Thr, and Tyr residues. However, only a few dozen phosphorylation sites in cyanobacteria are known, presenting a major obstacle for further understanding the regulatory roles of reversible phosphorylation in this group of bacteria. In this study, we carried out a global and site-specific phosphoproteomic analysis on the model cyanobacterium Synechococcus sp. PCC 7002. In total, 280 phosphopeptides and 410 phosphorylation sites from 245 Synechococcus sp. PCC 7002 proteins were identified through the combined use of protein/peptide prefractionation, TiO2 enrichment, and LC-MS/MS analysis. The identified phosphoproteins were functionally categorized into an interaction map and found to be involved in various biological processes such as two-component signaling pathway and photosynthesis. Our data provide the first global survey of phosphorylation in cyanobacteria by using a phosphoproteomic approach and suggest a wide-ranging regulatory scope of this modification. The provided data set may help reveal the physiological functions underlying Ser/Thr/Tyr phosphorylation and facilitate the elucidation of the entire signaling networks in cyanobacteria.
Use: pFind
2013 ICME International Conference on Complex Medical Engineering (CME)2013. Su, R et al.
Beijing Inst Technol, Sch Life Sci & Technol, Beijing 100081, Peoples R China.
ABSTRACT:Evidence shows that non-enzymatic glycation plays an important role in the development of diabetes, diabetes-related complications and neurodegenerative diseases. Mass spectrometric methods have been used for non-enzymatic glycation of proteins. At present, CID remains the major fragmentation method for peptide sequencing. In this study, we utilized synthesized peptide models to study fragmentation spectra of glycated peptides, which can be easily studied using ESI-MS. MS/MS fragmentation whose characteristics of glycated peptides will help for large-scale glycated proteomics analyses of complex plasma and tissue samples.
Use: pFind; pBuild; pLabel
Molecular Biosystems2012. Xie, LQ et al.
Fudan Univ, Shanghai Canc Ctr, Shanghai 200032, Peoples R China.
ABSTRACT:Electron transfer dissociation (ETD) is a useful and complementary activation method for peptide fragmentation in mass spectrometry. However, ETD spectra typically receive a relatively low score in the identifications of 2+ ions. To overcome this challenge, we, for the first time, systematically interrogated the benefits of combining ion charge enhancing methods (dimethylation, guanidination, m-nitrobenzyl alcohol (m-NBA) or Lys-C digestion) and differential search algorithms (Mascot, Sequest, OMSSA, pFind and X!Tandem). A simple sample (BSA) and a complex sample (AMJ2 cell lysate) were selected in benchmark tests. Clearly distinct outcomes were observed through different experimental protocol. In the analysis of AMJ2 cell lines, X!Tandem and pFind revealed 92.65% of identified spectra; m-NBA adduction led to a 5-10% increase in average charge state and the most significant increase in the number of successful identifications, and Lys-C treatment generated peptides carrying mostly triple charges. Based on the complementary identification results, we suggest that a combination of m-NBA and Lys-C strategies accompanied by X!Tandem and pFind can greatly improve ETD identification.
Use: pFind
Journal of Structural and Functional Genomics2013. Merkley, Eric D. et al.
Biological Sciences Division, Pacific Northwest, National Laboratory
ABSTRACT:Multiprotein complexes, rather than individual proteins, make up a large part of the biological macromolecular machinery of a cell. Understanding the structure and organization of these complexes is critical to understanding cellular function. Chemical cross-linking coupled with mass spectrometry is emerging as a complementary technique to traditional structural biology methods and can provide low-resolution structural information for a multitude of purposes, such as distance constraints in computational modeling of protein complexes. In this review, we discuss the experimental considerations for successful application of chemical cross-linking-mass spectrometry in biological studies and highlight three examples of such studies from the recent literature. These examples (as well as many others) illustrate the utility of a chemical cross-linking-mass spectrometry approach in facilitating structural analysis of large and challenging complexes.
Use: pLink
Skeletal Muscle2011. Mate, SE et al.
George Washington Univ, Dept Biochem & Mol Genet, IBS, Washington, DC USA.
ABSTRACT:Background: During development, the branchial mesoderm of Torpedo californica transdifferentiates into an electric organ capable of generating high voltage discharges to stun fish. The organ contains a high density of cholinergic synapses and has served as a biochemical model for the membrane specialization of myofibers, the neuromuscular junction (NMJ). We studied the genome and proteome of the electric organ to gain insight into its composition, to determine if there is concordance with skeletal muscle and the NMJ, and to identify novel synaptic proteins. Results: Of 435 proteins identified, 300 mapped to Torpedo cDNA sequences with >= 2 peptides. We identified 14 uncharacterized proteins in the electric organ that are known to play a role in acetylcholine receptor clustering or signal transduction. In addition, two human open reading frames, C1orf123 and C6orf130, showed high sequence similarity to electric organ proteins. Our profile lists several proteins that are highly expressed in skeletal muscle or are muscle specific. Synaptic proteins such as acetylcholinesterase, acetylcholine receptor subunits, and rapsyn were present in the electric organ proteome but absent in the skeletal muscle proteome. Conclusions: Our integrated genomic and proteomic analysis supports research describing a muscle-like profile of the organ. We show that it is a repository of NMJ proteins but we present limitations on its use as a comprehensive model of the NMJ. Finally, we identified several proteins that may become candidates for signaling proteins not previously characterized as components of the NMJ.
Use: pXtract
Bioinformatics2013. Jeong, K et al.
Univ Calif San Diego, Dept Elect & Comp Engn, San Diego, CA 92093 USA.
ABSTRACT:Motivation: Mass spectrometry (MS) instruments and experimental protocols are rapidly advancing, but de novo peptide sequencing algorithms to analyze tandem mass (MS/MS) spectra are lagging behind. Although existing de novo sequencing tools perform well on certain types of spectra [e.g. Collision Induced Dissociation (CID) spectra of tryptic peptides], their performance often deteriorates on other types of spectra, such as Electron Transfer Dissociation (ETD), Higher-energy Collisional Dissociation (HCD) spectra or spectra of non-tryptic digests. Thus, rather than developing a new algorithm for each type of spectra, we develop a universal de novo sequencing algorithm called UniNovo that works well for all types of spectra or even for spectral pairs (e.g. CID/ETD spectral pairs). UniNovo uses an improved scoring function that captures the dependences between different ion types, where such dependencies are learned automatically using a modified offset frequency function. Results: The performance of UniNovo is compared with PepNovo+, PEAKS and pNovo using various types of spectra. The results show that the performance of UniNovo is superior to other tools for ETD spectra and superior or comparable with others for CID and HCD spectra. UniNovo also estimates the probability that each reported reconstruction is correct, using simple statistics that are readily obtained from a small training dataset. We demonstrate that the estimation is accurate for all tested types of spectra (including CID, HCD, ETD, CID/ETD and HCD/ETD spectra of trypsin, LysC or AspN digested peptides).
Use: pNovo
BIOINFORMATICS2013. Chuang Li et al.
Univ Calif San Diego, Dept Elect & Comp Engn, San Diego, CA 92093 USA
ABSTRACT:Motivation: Mass spectrometry (MS) instruments and experimental protocols are rapidly advancing, but de novo peptide sequencing algorithms to analyze tandem mass (MS/MS) spectra are lagging behind. Although existing de novo sequencing tools perform well on certain types of spectra [e.g. Collision Induced Dissociation (CID) spectra of tryptic peptides], their performance often deteriorates on other types of spectra, such as Electron Transfer Dissociation (ETD), Higher-energy Collisional Dissociation (HCD) spectra or spectra of non-tryptic digests. Thus, rather than developing a new algorithm for each type of spectra, we develop a universal de novo sequencing algorithm called UniNovo that works well for all types of spectra or even for spectral pairs (e.g. CID/ETD spectral pairs). UniNovo uses an improved scoring function that captures the dependences between different ion types, where such dependencies are learned automatically using a modified offset frequency function.Results: The performance of UniNovo is compared with PepNovo+, PEAKS and pNovo using various types of spectra. The results show that the performance of UniNovo is superior to other tools for ETD spectra and superior or comparable with others for CID and HCD spectra. UniNovo also estimates the probability that each reported reconstruction is correct, using simple statistics that are readily obtained from a small training dataset. We demonstrate that the estimation is accurate for all tested types of spectra (including CID, HCD, ETD, CID/ETD and HCD/ETD spectra of trypsin, LysC or AspN digested peptides).
Use: pNovo
Proceedings of the National Academy of Sciences of the United States of America2012. Zhao, YM et al.
Natl Inst Biol Sci, Beijing 102206, Peoples R China.
ABSTRACT:Spermiogenesis is a series of poorly understood morphological, physiological and biochemical processes that occur during the transition of immotile spermatids into motile, fertilization-competent spermatozoa. Here, we identified a Serpin (serine protease inhibitor) family protein (As_SRP-1) that is secreted from spermatids during nematode Ascaris suum spermiogenesis (also called sperm activation) and we showed that As_SRP-1 has two major functions. First, As_SRP-1 functions in cis to supportmajor spermprotein (MSP)-based cytoskeletal assembly in the spermatid that releases it, thereby facilitating sperm motility acquisition. Second, As_SRP-1 released from an activated sperm inhibits, in trans, the activation of surrounding spermatids by inhibiting vas deferens-derived As_TRY-5, a trypsin-like serine protease necessary for sperm activation. Because vesicular exocytosis is necessary to create fertilization-competent sperm in many animal species, components released during this process might be more important modulators of the physiology and behavior of surrounding sperm than was previously appreciated.
Use: pNovo
Proteome Science2013. Cleveland, JP et al.
Univ S Carolina, Dept Comp Sci, Columbia, SC 29208 USA.
ABSTRACT:Independent of the approach used, the ability to correctly interpret tandem MS data depends on the quality of the original spectra. Even in the case of the highest quality spectra, the majority of spectral peaks can not be reliably interpreted. The accuracy of sequencing algorithms can be improved by filtering out such 'noise' peaks. Preprocessing MS/MS spectra to select informative ion peaks increases accuracy and reduces the processing time. Intuitively, the mix of informative versus non-informative peaks has a direct effect on the quality and size of the resulting candidate peptide search space. As the number of selected peaks increases, the corresponding search space increases exponentially. If we select too few peaks then the ion-ladder interpretation of the spectrum will contain gaps that can only be explained by permutations of combinations of amino acids. This will result in a larger candidate peptide search space and poorer quality candidates. The dependency that peptide sequencing accuracy has on an initial peak selection regime makes this preprocessing step a crucial facet of any approach, whether de novo or not, to MS/MS spectra interpretation. We have developed a novel approach to address this problem. Our approach uses a staged neural network to model ion fragmentation patterns and estimate the posterior probability of each ion type. Our method improves upon other preprocessing techniques and shows a significant reduction in the search space for candidate peptides without sacrificing candidate peptide quality.
Use: pNovo