Sample Preparation Methods for Mass Spectrometric Peptide Mapping Directly from 2-DE Gels

Jensen, Ole Mejlhede; Wilm, Matthias; Shevchenko, Andrej; Mann, Matthias

doi:10.1385/1-59259-584-7:513

Cited by 213 publications

(223 citation statements)

References 37 publications

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“…Protein spots were excised and digested with trypsin by modification of the method of Jensen et al (34). The excised gel pieces were incubated for 15 min in 100 mM NH 4 HCO 3 and 50% acetonitrile and were dried by vacuum centrifugation.…”

Section: Identification Of Proteins By Mass Spectrometrymentioning

confidence: 99%

Myeloid-Related Protein-14 Is a p38 MAPK Substrate in Human Neutrophils

Lominadze

Rane

Merchant

et al. 2005

The Journal of Immunology

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The targets of the p38 MAPK pathway that mediate neutrophil functional responses are largely unknown. To identify p38 MAPK targets, a proteomic approach was applied in which recombinant active p38 MAPK and [32P]ATP were added to lysates from unstimulated human neutrophils. Proteins were separated by two-dimensional gel electrophoresis, and phosphoproteins were visualized by autoradiography and identified by MALDI-TOF. Myeloid-related protein-14 (MRP-14) was identified as a candidate p38 MAPK substrate. MRP-14 phosphorylation by p38 MAPK was confirmed by an in vitro kinase reaction using purified MRP-14/MRP-8 complexes. The site of MRP-14 phosphorylation by p38 MAPK was identified by tandem mass spectrometry and site-directed mutagenesis to be Thr113. MRP-14 phosphorylation by p38 MAPK in intact neutrophils was confirmed by [32P]orthophosphate loading, followed by fMLP stimulation in the presence and absence of a p38 MAPK inhibitor, SB203580. Confocal microscopy of Triton X-100 permeabilized neutrophils showed that a small amount of MRP-14 was associated with cortical F-actin in unstimulated cells. fMLP stimulation resulted in a p38 MAPK-dependent increase in MRP-14 staining at the base of lamellipodia. By immunoblot analysis, MRP-14 was present in plasma membrane/secretory vesicle fractions and gelatinase and specific granules, but not in azurophil granules. The amount of MRP-14 associated with plasma membrane/secretory vesicle and gelatinase granule fractions increased after fMLP stimulation in a p38 MAPK-dependent manner. Direct phosphorylation of the MRP-14/MRP-8 complex by p38 MAPK increased actin binding in vitro by 2-fold. These results indicate that MRP-14 is a potential mediator of p38 MAPK-dependent functional responses in human neutrophils.

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Section: Identification Of Proteins By Mass Spectrometrymentioning

confidence: 99%

Myeloid-Related Protein-14 Is a p38 MAPK Substrate in Human Neutrophils

Lominadze

Rane

Merchant

et al. 2005

The Journal of Immunology

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“…MALDI-TOF MS produces spectra by the measurement of masses of intact peptides and identifies proteins by the correlation of these masses with theoretically calculated peptides from database entries (see [5] for tutorial). The second type of analysis, tandem mass spectrometry (MS/MS), produces patterns of peptide fragments that can be interpreted and/or correlated to database entries in a number of ways (for tutorial on nanoelectropsray MS/MS see [6], for nanoLC MS/MS [7], and [8] for generic review).…”

Section: Cross-species Protein Identification By Msmentioning

confidence: 99%

Expanding the organismal scope of proteomics: Cross‐species protein identification by mass spectrometry and its implications

2003

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“…In a one-dimensional (1D) LC-MS/MS experiment, peptide mixtures are typically separated only by hydrophobicity by passage over a column packed with non-polar stationary phase. Thus, the number of proteins identified using 1D-LC-MS/MS is directly dependent on the efficiency of peptide separation prior to introduction into the mass spectrometer (Jensen et al, 1999). In LC-MS/MS experiments, ESI is the dominant method of ionization.…”

Section: Lc-ms/msmentioning

confidence: 99%

“…Peak lists generated from the fragment ion masses in tandem mass spectra are then searched against a protein database to determine the amino acid sequence of the peptides in the complex mixture. The assignment of the sequenced peptides to a given protein is the means by which protein identification is accomplished (Jensen et al, 1999).…”

Section: Lc-ms/msmentioning

confidence: 99%