分享一个蛋白质组学的方法,经多家实验室验证# Biology - 生物学
f*K
1 楼
在博后期间,合成一种Cys特异性的phosphanate tag,用于Cys Proteome, Cys PTMs
,以及the cross-talk between Cys PTMs and phosphorylation研究应用。方法具有
高度的特异性,高效性,已经多家独立实验室验证。
关于方法意义,引用reviewer comment:
1. To my knowledgy, the CysPAT strategy is the first method to
simultaneously
measure cysteine proteome and protein phosphorylation from the same sample,
which is the major novelty of this study.
相关tag合成方法及应用方法,已经申请美国专利。
文章在MCP发表:http://www.ncbi.nlm.nih.gov/pubmed/27281782
欢迎大家讨论使用。
Abstract:
Cysteine is a rare and conserved amino acid involved in most cellular
functions. The thiol group of cysteine can be subjected to diverse oxidative
modifications that regulate many physio-pathological states. In the present
work, a Cysteine-specific Phosphonate Adaptable Tag (CysPAT) was
synthesized to selectively label cysteine-containing peptides (Cys peptides)
followed by their enrichment with titanium dioxide (TiO2) and subsequent
mass spectrometric analysis. The CysPAT strategy was developed using a
synthetic peptide, a standard protein and subsequently the strategy was
applied to protein lysates from Hela cells, achieving high specificity and
enrichment efficiency. In particular, for Cys proteome analysis, the method
led to the identification of 7509 unique Cys peptides from 500ug of HeLa
cell lysate starting material. Furthermore, the method was developed to
simultaneously enrich Cys peptides and phosphorylated peptides. This
strategy was applied to SILAC labelled Hela cells subjected to 5 min
epidermal growth factor (EGF) stimulation. In total, 10440 unique reversibly
modified Cys peptides (3855 proteins) and 7339 unique phosphopeptides (2234
proteins) were simultaneously identified from 250 ug starting material.
Significant regulation was observed in both phosphorylation and reversible
Cys modification of proteins involved in EGFR signaling. Our data indicates
that EGF stimulation can activate the well-known phosphorylation of EGFR and
downstream signaling molecules, such as mitogen-activated protein kinases (
MAPK1 and MAPK3), however, it also leads to substantial modulation of
reversible cysteine modifications in numerous proteins. Several protein
tyrosine phosphatases (PTPs) showed a reduction of the catalytic Cys site in
the conserved putative phosphatase HC(X)5R motif indicating an activation
and subsequent de-phosphorylation of proteins involved in the EGF signaling
pathway. Overall, the CysPAT strategy is a straight forward, easy and
promising method for studying redox proteomics and the simultaneous
enrichment strategy offers an excellent solution for characterization of
cross-talk between phosphorylation and redox induced reversible cysteine
modifications.
,以及the cross-talk between Cys PTMs and phosphorylation研究应用。方法具有
高度的特异性,高效性,已经多家独立实验室验证。
关于方法意义,引用reviewer comment:
1. To my knowledgy, the CysPAT strategy is the first method to
simultaneously
measure cysteine proteome and protein phosphorylation from the same sample,
which is the major novelty of this study.
相关tag合成方法及应用方法,已经申请美国专利。
文章在MCP发表:http://www.ncbi.nlm.nih.gov/pubmed/27281782
欢迎大家讨论使用。
Abstract:
Cysteine is a rare and conserved amino acid involved in most cellular
functions. The thiol group of cysteine can be subjected to diverse oxidative
modifications that regulate many physio-pathological states. In the present
work, a Cysteine-specific Phosphonate Adaptable Tag (CysPAT) was
synthesized to selectively label cysteine-containing peptides (Cys peptides)
followed by their enrichment with titanium dioxide (TiO2) and subsequent
mass spectrometric analysis. The CysPAT strategy was developed using a
synthetic peptide, a standard protein and subsequently the strategy was
applied to protein lysates from Hela cells, achieving high specificity and
enrichment efficiency. In particular, for Cys proteome analysis, the method
led to the identification of 7509 unique Cys peptides from 500ug of HeLa
cell lysate starting material. Furthermore, the method was developed to
simultaneously enrich Cys peptides and phosphorylated peptides. This
strategy was applied to SILAC labelled Hela cells subjected to 5 min
epidermal growth factor (EGF) stimulation. In total, 10440 unique reversibly
modified Cys peptides (3855 proteins) and 7339 unique phosphopeptides (2234
proteins) were simultaneously identified from 250 ug starting material.
Significant regulation was observed in both phosphorylation and reversible
Cys modification of proteins involved in EGFR signaling. Our data indicates
that EGF stimulation can activate the well-known phosphorylation of EGFR and
downstream signaling molecules, such as mitogen-activated protein kinases (
MAPK1 and MAPK3), however, it also leads to substantial modulation of
reversible cysteine modifications in numerous proteins. Several protein
tyrosine phosphatases (PTPs) showed a reduction of the catalytic Cys site in
the conserved putative phosphatase HC(X)5R motif indicating an activation
and subsequent de-phosphorylation of proteins involved in the EGF signaling
pathway. Overall, the CysPAT strategy is a straight forward, easy and
promising method for studying redox proteomics and the simultaneous
enrichment strategy offers an excellent solution for characterization of
cross-talk between phosphorylation and redox induced reversible cysteine
modifications.