Large-scale proteomics will play a critical role in the rapid display, identification and validation of new protein targets, and elucidation of the underlying molecular events that are associated with disease development, progression and severity. However, because the proteome of most organisms are significantly more complex than the genome, the comprehensive analysis of protein expression changes will require an analytical effort beyond the capacity of standard laboratory equipment. We describe the first high-throughput proteomic analysis of human breast infiltrating ductal carcinoma (IDCA) using OCT (optimal cutting temperature) embedded biopsies, two-dimensional difference gel electrophoresis (2-D DIGE) technology and a fully automated spot handling workstation. Total proteins from four breast IDCAs (Stage I, IIA, IIB and IIIA) were individually compared to protein from non-neoplastic tissue obtained from a female donor with no personal or family history of breast cancer. We detected differences in protein abundance that ranged from 14.8% in stage I IDCA versus normal, to 30.6% in stage IIB IDCA versus normal. A total of 524 proteins that showed > or = three-fold difference in abundance between IDCA and normal tissue were picked, processed and identified by mass spectrometry. Out of the proteins picked, approximately 80% were unambiguously assigned identities by matrix-assisted laser desorbtion/ionization-time of flight mass spectrometry or liquid chromatography-tandem mass spectrometry in the first pass. Bioinformatics tools were also used to mine databases to determine if the identified proteins are involved in important pathways and/or interact with other proteins. Gelsolin, vinculin, lumican, alpha-1-antitrypsin, heat shock protein-60, cytokeratin-18, transferrin, enolase-1 and beta-actin, showed differential abundance between IDCA and normal tissue, but the trend was not consistent in all samples. Out of the proteins with database hits, only heat shock protein-70 (more abundant) and peroxiredoxin-2 (less abundant) displayed the same trend in all the IDCAs examined. This preliminary study demonstrates quantitative and qualitative differences in protein abundance between breast IDCAs and reveals 2-D DIGE portraits that may be a reflection of the histological and pathological status of breast IDCA.
Cysteine phosphorylation has recently been discovered in both prokaryotic and eukaryotic systems, and is thought to play crucial roles in signaling and regulation of cellular responses. This article explores the topics of chemical stability of this type of structural modification and the resulting issues regarding affinity enrichment of S-phosphopeptides and their mass spectrometry-based detection in the course of general proteomics studies. Together, this work suggests that the current advances in phosphoproteomic methodologies provide adequate tools for investigating protein cysteine phosphorylation and appear to be immediately available for practical implementation. The article provides useful information necessary for designing experiments in the emerging cysteine phosphoproteomics. The examples of methodological proposals for S-linked phosphorylation detection are included herein in order to stimulate development of new approaches by the phosphoproteomic community.
The regulatory role of protein cysteine phosphorylation is an under-researched area. The difficulty of accessing reference S-phosphorylated peptides (pCys-peptides) hampers progress in MS-driven cysteine phosphoproteomics, which requires targeted analytical procedures. This work describes an uncomplicated process for the conversion of disulfide-bridged protein into a complex model mixture of combinatorially modified peptides. Hen egg-white lysozyme was reduced with tris(2carboxyethyl)phosphine (TCEP) followed by alkylation of cysteine with (3-acrylamidopropyl)trimethyl-ammonium chloride (APTA) and subsequent beta-elimination in aqueous Ba(OH) 2 to yield modified polypeptides containing multiple dehydroalanine (Dha) residues. The conjugate addition of thiophosphoric acid to Dha residues followed by trypsinolysis led to numerous D/L phosphocysteine-containing peptides, which were identified by higher-energy collisional-dissociation tandem mass spectrometry (HCD-MS/MS). Our results show that some pCys-peptides produce prominent neutral losses of 80 Da, 98 Da and a weak 116 Da loss. These are similar to the neutral-loss triplets generated by phosphohistidine peptides.
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