Dataset Information

Alpha-lytic proteases

ABSTRACT: S. pombe proteome digested separately by four proteases and analyzed with an LTQ-Orbitrap Velos with a data-dependent decision tree method.

PROVIDER: MSV000078516 | MassIVE | Mon Dec 23 16:44:00 GMT 2013

REPOSITORIES: MassIVE

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Expanding proteome coverage with orthogonal-specificity α-lytic proteases.

Meyer Jesse G JG Kim Sangtae S Maltby David A DA Ghassemian Majid M Bandeira Nuno N Komives Elizabeth A EA

Molecular & cellular proteomics : MCP 20140114 3

Bottom-up proteomics studies traditionally involve proteome digestion with a single protease, trypsin. However, trypsin alone does not generate peptides that encompass the entire proteome. Alternative proteases have been explored, but most have specificity for charged amino acid side chains. Therefore, additional proteases that improve proteome coverage through cleavage at sequences complementary to trypsin's may increase proteome coverage. We demonstrate the novel application of two proteases f ...[more]

PMID: 24425750

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Project description:Proteases and protease inhibitors have been analyzed in the non-orthodox Quercus ilex forest tree by using in silico and wet approaches. The in silico analysis showed that the species-specific transcriptome database included 2240 and 97 transcripts annotated as, respectively, proteases and protease inhibitors, with members of the different families according to the MEROPS protease database (http://www.merops.ac.uk) classification, mostly of serine- and metallo-type. Data for Q. ilex are compared with those previously published for other Quercus species, namely, Q. suber, Q. lobata, and Q. robur. A second approach was performed to assess the evolution of proteases and protease inhibitors along with seed germination in cotyledon and embryo tissues by determining changes at proteomics and activity levels, the last by mean of in vitro and in gel assays. Shotgun (LC-MSMS) analysis of acorn embryos and cotyledons of three developmental stages (NV, non-viable; T1, mature; and T3 germinated) allowed the identification of a total of 177 proteases and 12 protease inhibitors. In vitro protease activity was determined by using azocasein as substrate; the total activity was higher in cotyledons than embryos, showing the latter significant differences throughout germination. Gel protease activity assays showed common and differential bands between tissues and developmental stages. Shotgun analysis of 10 differential activity bands supported the results obtained from the whole proteome analysis, with some proteins highly represented in the same tissues and stages by both strategies. In overall, the combination of shotgun proteomics and protease activity approaches allowed the identification of tissue-specific (such as cysteine protease inhibitors), and stage-specific proteins (such as those related to storage proteins mobilization), with others being present in all the analyzed organs. Some of the proteases and protease inhibitors differentially identified between tissues and stages in this study could be used as indicators of seed quality and viability.

Dataset Information

Alpha-lytic proteases

Publications

Expanding proteome coverage with orthogonal-specificity α-lytic proteases.

Similar Datasets

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