pFind Studio: a computational solution for mass spectrometry-based proteomics
2017
Analytical chemistry2017. Horton, AP et al.
Univ Texas Austin, Dept Mol Biosci, Ctr Syst & Synthet Biol, Austin, TX 78712 USA.
ABSTRACT:We describe a strategy for de novo peptide sequencing based on matched pairs of tandem mass spectra (MS/MS) obtained by collision induced dissociation (CID) and 351 nm ultraviolet photodissociation (UVPD). Each precursor ion is isolated twice with the mass spectrometer switching between CID and UVPD activation modes to obtain a complementary MS/MS pair. To interpret these paired spectra, we modified the UVnovo de novo sequencing software to automatically learn from and interpret fragmentation spectra, provided a representative set of training data. This machine learning procedure, using random forests, synthesizes information from one or multiple complementary spectra, such as the CID/UVPD pairs, into peptide fragmentation site predictions. In doing so, the burden of fragmentation model definition shifts from programmer to machine and opens up the model parameter space for inclusion of nonobvious features and interactions. This spectral synthesis also serves to transform distinct types of spectra into a common representation for subsequent activation-independent processing steps. Then, independent from precursor activation constraints, UVnovo's de novo sequencing procedure generates and scores sequence candidates for each precursor. We demonstrate the combined experimental and computational approach for de novo sequencing using whole cell E. coli lysate. In benchmarks on the CID/UVPD data, UVnovo assigned correct full-length sequences to 83% of the spectral pairs of doubly charged ions with high-confidence database identifications. Considering only top-ranked de novo predictions, 70% of the pairs were deciphered correctly. This de novo sequencing performance exceeds that of PEAKS and PepNovo on the CID spectra and that of UVnovo on CID or UVPD spectra alone. As presented here, the methods for paired CID/UVPD spectral acquisition and interpretation constitute a powerful workflow for high-throughput and accurate de novo peptide sequencing.
Use: pNovo
Analytical chemistry2017. Horton, AP et al.
Univ Texas Austin, Dept Mol Biosci, Ctr Syst & Synthet Biol, Austin, TX 78712 USA.
ABSTRACT:We describe a strategy for de novo peptide sequencing based on matched pairs of tandem mass spectra (MS/MS) obtained by collision induced dissociation (CID) and 351 nm ultraviolet photodissociation (UVPD). Each precursor ion is isolated twice with the mass spectrometer switching between CID and UVPD activation modes to obtain a complementary MS/MS pair. To interpret these paired spectra, we modified the UVnovo de novo sequencing software to automatically learn from and interpret fragmentation spectra, provided a representative set of training data. This machine learning procedure, using random forests, synthesizes information from one or multiple complementary spectra, such as the CID/UVPD pairs, into peptide fragmentation site predictions. In doing so, the burden of fragmentation model definition shifts from programmer to machine and opens up the model parameter space for inclusion of nonobvious features and interactions. This spectral synthesis also serves to transform distinct types of spectra into a common representation for subsequent activation-independent processing steps. Then, independent from precursor activation constraints, UVnovo's de novo sequencing procedure generates and scores sequence candidates for each precursor. We demonstrate the combined experimental and computational approach for de novo sequencing using whole cell E. coli lysate. In benchmarks on the CID/UVPD data, UVnovo assigned correct full-length sequences to 83% of the spectral pairs of doubly charged ions with high-confidence database identifications. Considering only top-ranked de novo predictions, 70% of the pairs were deciphered correctly. This de novo sequencing performance exceeds that of PEAKS and PepNovo on the CID spectra and that of UVnovo on CID or UVPD spectra alone. As presented here, the methods for paired CID/UVPD spectral acquisition and interpretation constitute a powerful workflow for high-throughput and accurate de novo peptide sequencing.
Use: pNovo
Atherosclerosis2017. Sun, JT et al.
Karolinska Inst, Unit Immunol & Chron Dis, Inst Environm Med, Stockholm, Sweden.
ABSTRACT:Background and aims: IgM antibodies against phosphorylcholine (anti-PC) are negatively associated with atherosclerosis, cardiovascular disease (CVD) and systemic lupus erythematosus (SLE), where the risk of CVD and atherosclerosis is high. We here study the effects of IgM anti-PC immune regulation. Methods: Mononuclear leukocytes were isolated from peripheral blood (PBMC) obtained from healthy blood donors, six SLE patients with age-and sex-matched controls, and symptom-giving human atherosclerotic plaques. The proportion of Th17 (CD4(+)CCR6(+)) and Treg (CD4(+)CD25(+)CD127(dim/-)) cells was determined by flow cytometry in CD4(+)T cells after 6 days of culture with Th17 or Treg-polarizing cytokines, with PMA and Ionomycin stimulation. IgM anti-PC were extracted from total IgM, with flowthrough IgM as controls. Dendritic cells (DC) were differentiated from PBMC. Antibody peptide/protein characterization was done by a proteomics de novo sequencing approach. Results: IgM anti-PC increased significantly the proportion of Tregs from healthy donors, SLE patients and atherosclerotic plaque cells while control antibodies did not. T cells from SLE patients had a significantly lower proportion of Tregs and a higher proportion of Th17 cells as compared to matched controls. IgM anti-PC, but not control antibodies, significantly reduced the production of IL-17 and TNF-alpha in cell cultures from SLE patients and atherosclerotic plaque cells. IgM anti-PC interacted with CD40 and kept DCs in an immature stage, potentially being tolerogenic. We observed differences in the IgM peptide expression levels in anti-PC compared to control antibodies. Conclusions: IgM anti-PC promote polarization of Tregs, which could represent a novel protective mechanism in atherosclerosis and autoimmune conditions as SLE. (C) 2017 Published by Elsevier Ireland Ltd.
Use: pNovo
Journal of Proteome Research2017. Blank-Landeshammer, B et al.
ISAS eV, Leibniz Inst Analyt Wissensch, D-44139 Dortmund, Germany.
ABSTRACT:Complex mass spectrometry based proteomics data sets are mostly analyzed by protein database searches. While this approach performs considerably well for sequenced organisms, direct inference of peptide sequences from tandem mass spectra, i.e., de novo peptide sequencing, oftentimes is the only way to obtain information when protein databases are absent. However, available algorithms suffer from drawbacks such as lack of validation and often high rates of false positive hits (FP). Here we present a simple method of combining results from commonly available de novo peptide sequencing algorithms, which in conjunction with minor tweaks in data acquisition ensues lower empirical FDR compared to the analysis using single algorithms. Results were validated using state-of-the art database search algorithms as well specifically synthesized reference peptides. Thus, we could increase the number of PSMs meeting a stringent FDR of 5% more than 3-fold compared to the single best de novo sequencing algorithm alone, accounting for an average of 11 120 PSMs (combined) instead of 3476 PSMs (alone) in triplicate 2 h LC-MS runs of tryptic HeLa digestion.
Use: pNovo
Journal of proteome research2017. Ma, YH et al.
Scripps Res Inst, Dept Chem Physiol & Mol & Cellular Neurobiol, La Jolla, CA 92037 USA.
ABSTRACT:Here we describe a new strategy, HILAQ (Heavy Isotope Labeled Azidohomoalanine Quantification), to rapidly quantify the molecular vulnerability profile to oxytosis, which is an oxidative stress-induced programed cell death pathway that has been reported to be involved in aging and neurodegenerative diseases. HILAQ was able to quantify 1962 newly synthesized proteins (NSPs) after 1 h of pulse labeling in HEK293T cell line, while 353 proteins were quantified using the previously published QuaNCAT protocol. HILAQ was successfully applied to the HT22 oxytosis model. 226 proteins were found to have a two-fold change in abundance, and 108 proteins were enriched in the cell death pathway, demonstrating the utility of HT22 cells as a tool to study the molecular details of cell death involved in neurodegenerative diseases. The HILAQ strategy simplifies the analysis of newly synthesized proteomes through the use of isobaric labels and achieves higher sensitivity than previously published methods.
Use: pQuant
Journal of proteome research2017. Ma, YH et al.
Scripps Res Inst, Dept Chem Physiol & Mol & Cellular Neurobiol, La Jolla, CA 92037 USA.
ABSTRACT:Here we describe a new strategy, HILAQ (Heavy Isotope Labeled Azidohomoalanine Quantification), to rapidly quantify the molecular vulnerability profile to oxytosis, which is an oxidative stress-induced programed cell death pathway that has been reported to be involved in aging and neurodegenerative diseases. HILAQ was able to quantify 1962 newly synthesized proteins (NSPs) after 1 h of pulse labeling in HEK293T cell line, while 353 proteins were quantified using the previously published QuaNCAT protocol. HILAQ was successfully applied to the HT22 oxytosis model. 226 proteins were found to have a two-fold change in abundance, and 108 proteins were enriched in the cell death pathway, demonstrating the utility of HT22 cells as a tool to study the molecular details of cell death involved in neurodegenerative diseases. The HILAQ strategy simplifies the analysis of newly synthesized proteomes through the use of isobaric labels and achieves higher sensitivity than previously published methods.
Use: pQuant
Molecular Plant2017. Bu, Shuo-Lei et al.
Carnegie Inst Sci, Dept Plant Biol, 290 Panama St, Stanford, CA 94305 USA; Hebei Normal Univ, Hebei Collaborat Innovat Ctr Cell Signaling, Hebei Key Lab Mol & Cellular Biol, Coll Life Sci, Shijiazhuang 050024, Hebei, Peoples R China
ABSTRACT:ABA induces the phosphorylation of three basic helix-loop-helix (bHLH) transcription factors, called AKSs (ABA-responsive kinase substrates; AKS1,AKS2, and AKS3). The unphosphorylated AKSs facilitate stomatal opening through promoting the transcription of genes encoding inwardly rectifying K+ channels (Takahashi et al., 2013). AKS1 and AKS3 are also regulators of flowering (Ito et al., 2012). However, the kinases and phosphatases that directly control the phosphorylation status of AKSs in vivo have not been fully characterized. Here, our proteomic analyses provide evidence supporting that AKSs are phosphorylated by GSK3 kinases and dephosphorylated by protein phosphatase 2A (PP2A).
Use: pQuant
Immunological Investigations2017. Takuse, Y et al.
Osaka Univ, Grad Sch Med, Dept Biomed Informat, Div Hlth Sci, Yamadaoka 1-7, Suita, Osaka 5650871, Japan.
ABSTRACT:Interleukin (IL)-10 is known to suppress inflammation in autoimmune diseases. IL-10 can be regulated by miRNAs. To elucidate the involvement of miRNAs that regulate IL-10 expression with the pathogenesis of autoimmune thyroid disease (AITD), we examined the expression levels of hsa-miR-27a-3p, hsa-miR-98-5p, hsa-miR-106a-5p, and hsamiR- 223-3p in peripheral blood mononuclear cells (PBMCs) from 43 patients with Graves' disease (GD), 38 patients with Hashimoto's disease (HD), and 21 healthy volunteers. We evaluated the association between the expression levels of four miRNAs and intracellular expression of IL-10 in PBMCs from 11 healthy volunteers. We also genotyped MIR27A rs895819 G/A and MIR106A rs3747440 C/G polymorphisms, which may be related to the expression of these miRNAs in 141 patients with GD, 178 patients with HD, and 84 healthy volunteers. The expression level of hsa-miR-106a-5p was significantly higher in patients with intractable GD than in those with GD in remission (p = 0.0113). The expression level of hsa-miR-223-3p was significantly lower in GD than in HD and lower in patients with intractable GD than in healthy volunteers (p = 0.0094, 0.0340). We found a negative correlation between the expression levels of hsa-miR-98-5p and the proportions of IL-10(+) cells in stimulated PBMCs from healthy volunteers (p = 0.0092). The G allele of the MIR27A polymorphism was significantly more frequent in patients with mild HD than in healthy volunteers (p = 0.0432). In conclusion, the expression levels of hsa-miR106a- 5p and hsa-miR-223-3p were associated with the pathogenesis of AITDs. hsa-miR-98-5p may negatively regulate the expression of IL-10. The functional polymorphism of MIR27A was associated with HD severity.
Use: pMatch
Nature Methods2017. Park, J et al.
Pacific Northwest Natl Lab, Biol Sci Div, Richland, WA 99354 USA.
ABSTRACT:Top-down proteomics, the analysis of intact proteins in their endogenous form, preserves valuable information about post-translation modifications, isoforms and proteolytic processing. The quality of top-down liquid chromatography-tandem MS (LC-MS/MS) data sets is rapidly increasing on account of advances in instrumentation and sample-processing protocols. However, top-down mass spectra are substantially more complex than conventional bottom-up data. New algorithms and software tools for confident proteoform identification and quantification are needed. Here we present Informed-Proteomics, an open-source software suite for top-down proteomics analysis that consists of an LC-MS feature-finding algorithm, a database search algorithm, and an interactive results viewer. We compare our tool with several other popular tools using human-in-mouse xenograft luminal and basal breast tumor samples that are known to have significant differences in protein abundance based on bottom-up analysis.
Use: pTop