Global identification of protein kinase substrates by protein microarray analysis

Mok, Janine; Im, Hogune; Snyder, M

doi:10.1038/nprot.2009.194

Cited by 46 publications

(64 citation statements)

References 26 publications

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“…Purified recombinant yeast proteins were printed on the nitrocellulose coated glass slides as previously described (13). Each microarray contains 4,800 fulllength GST fusion proteins spotted in duplicate (14,15).…”

Section: Protein Microarray Screen and Data Analysismentioning

confidence: 99%

Evaluating Common Humoral Responses against Fungal Infections with Yeast Protein Microarrays

Coelho

Clemons

et al. 2015

J. Proteome Res.

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We profiled the global immunoglobulin response against fungal infection by using yeast protein microarrays. Groups of CD-1 mice were infected systemically with human fungal pathogens (Coccidioides posadasii, Candida albicans, or Paracoccidioides brasiliensis) or inoculated with PBS as a control. Another group was inoculated with heat-killed yeast (HKY) of Saccharomyces cerevisiae. After 30 days, serum from mice in the groups were collected and used to probe S. cerevisiae protein microarrays containing 4800 full-length glutathione S-transferase (GST)-fusion proteins. Antimouse IgG conjugated with Alexafluor 555 and anti-GST antibody conjugated with Alexafluor 647 were used to detect antibody-antigen interactions and the presence of GST-fusion proteins, respectively. Serum after infection with C. albicans reacted with 121 proteins: C. posadasii, 81; P. brasiliensis, 67; and after HKY, 63 proteins on the yeast protein microarray, respectively. We identified a set of 16 antigenic proteins that were shared across the three fungal pathogens. These include retrotransposon capsid proteins, heat shock proteins, and mitochondrial proteins. Five of these proteins were identified in our previous study of fungal cell wall by mass spectrometry (Ann. N. Y. Acad. Sci. 2012, 1273, 44-51). The results obtained give a comprehensive view of the immunological responses to fungal infections at the proteomic level. They also offer insight into immunoreactive protein commonality among several fungal pathogens and provide a basis for a panfungal vaccine.

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Section: Protein Microarray Screen and Data Analysismentioning

confidence: 99%

Evaluating Common Humoral Responses against Fungal Infections with Yeast Protein Microarrays

Coelho

Clemons

et al. 2015

J. Proteome Res.

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“…Early studies in yeast were mostly aimed at identifying kinase substrates using in vitro kinase assays (6), whereas recent technological advances have enabled proteome-wide mapping of phosphorylation sites in vivo (7). Systems-level studies monitoring growth (1,8) or changes in the abundance of transcripts (9) or protein phosphorylation (7) in yeast have resolved the effect of deletion or overexpression of individual members of a network of greater than 130 kinases and 50 phosphatases.…”

Section: Introductionmentioning

confidence: 99%

Large-scale functional analysis of the roles of phosphorylation in yeast metabolic pathways

et al. 2014

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Protein phosphorylation is a widespread posttranslational modification that regulates almost all cellular functions. To investigate the large number of phosphorylation events with unknown functions, we monitored the concentrations of several hundred intracellular metabolites in Saccharomyces cerevisiae yeast strains with deletions of 118 kinases or phosphatases. Whereas most deletion strains had no detectable difference in growth compared to wild-type yeast, two-thirds of deletion strains had alterations in metabolic profiles. For about half of the kinases and phosphatases encoded by the deleted genes, we inferred specific regulatory roles on the basis of knowledge about the affected metabolic pathways. We demonstrated that the phosphatase Ppq1 was required for metal homeostasis. Combining metabolomic data with published phosphoproteomic data in a stoichiometric model enabled us to predict functions for phosphorylation in the regulation of 47 enzymes. Overall, we provided insights and testable predictions covering greater than twice the number of known phosphorylated enzymes in yeast, suggesting extensive phosphorylation-dependent regulation of yeast metabolism.

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“…Protease C and protein A Cterminal fusion tags were removed through the use of sitedirected ligase independent mutagenesis (SLIM) as described by Chiu et al [36] using the primers shown in Table S-1. The plasmids were then transformed into wildtype yeast cells using the LiAc method [37] and proteins were overexpressed as described by Gelperin et al [38] and Mok et al [39]. Five mL of cells were harvested, resuspended in 1 mL of induction media and methylated in vivo by incubating in 50 μM SAM for 90 min.…”

Section: Overexpression and In Vivo Methylation Of Npl3mentioning

confidence: 99%

Enhanced Methylarginine Characterization by Post-Translational Modification-Specific Targeted Data Acquisition and Electron-Transfer Dissociation Mass Spectrometry

Hart‐Smith

Low

Erce

et al. 2012

J. Am. Soc. Mass Spectrom.

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When localizing protein post-translational modifications (PTMs) using liquid-chromatography (LC)-tandem mass spectrometry (MS/MS), existing implementations are limited by inefficient selection of PTM-carrying peptides for MS/MS, particularly when PTM site occupancy is sub-stoichiometric. The present contribution describes a method by which peptides carrying specific PTMs of interest-in this study, methylarginines-may be selectively targeted for MS/MS: peptide features are extracted from high mass accuracy single-stage MS data, searched against theoretical PTM-carrying peptide masses, and matching features are subjected to targeted data acquisition LC-MS/MS. Using trypsin digested Saccharomyces cerevisiae Npl3, in which evidence is presented for 18 methylarginine sites-17 of which fall within a glycine-arginine-rich (GAR) domain spanning G120 amino acids-it is shown that this approach outperforms conventional data dependent acquisition (DDA): when applied to a complex protein mixture featuring in vivo methylated Npl3, 95 % more (P=0.030) methylargininecarrying peptides are selected for MS/MS than DDA, leading to an 86 % increase (P=0.044) in the number of methylated peptides producing Mascot ion scores ≥20 following electron-transfer dissociation (ETD). Notably, significantly more low abundance arginine methylated peptides (maximum ion intensities G6×10 4 cps) are selected for MS/MS using this approach relative to DDA (50 % more in a digest of purified in vitro methylated Npl3). It is also demonstrated that relative to collision-induced dissociation (CID), ETD facilitates a 586 % increase (P=0.016) in average Mascot ion scores of methylarginine-carrying peptides. The present PTM-specific targeted data acquisition approach, though described using methylarginine, is applicable to any ionizable PTM of known mass.

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Global identification of protein kinase substrates by protein microarray analysis

Cited by 46 publications

References 26 publications

Evaluating Common Humoral Responses against Fungal Infections with Yeast Protein Microarrays

Evaluating Common Humoral Responses against Fungal Infections with Yeast Protein Microarrays

Large-scale functional analysis of the roles of phosphorylation in yeast metabolic pathways

Enhanced Methylarginine Characterization by Post-Translational Modification-Specific Targeted Data Acquisition and Electron-Transfer Dissociation Mass Spectrometry

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