Mass Spectrometric Characterization of Protein Structure Details Refines the Proteome Signature for Invasive Ductal Breast Carcinoma

Abstract

Early diagnosis as well as individualized therapies are necessary to reduce the mortality of breast cancer, and personalized patient care strategies rely on novel prognostic or predictive factors. In this study, with six breast cancer patients, 2D gel analysis was applied for studying protein expression differences in order to distinguish invasive ductal breast carcinoma, the most frequent breast tumor subtype, from control samples. In total, 1203 protein spots were assembled in a 2D reference gel. Differentially abundant spots were subjected to peptide mass fingerprinting for protein identification. Twenty proteins with their corresponding 38 differentially expressed 2D gel spots were contained in our previously reported proteome signature, suggesting that distinct protein forms were contributing. In-depth MS/MS measurements enabled analyses of protein structure details of selected proteins. In protein spots that significantly contributed to our signature, we found that glyceraldehyde-3-phosphate dehydrogenase was N-terminally truncated, pyruvate kinase M2 and nucleoside diphosphate kinase A but not other isoforms of these proteins were of importance, and nucleophosmin phosphorylation at serine residues 106 and 125 were clearly identified. Principle component analysis and hierarchical clustering with normalized quantitative data from the 38 spots resulted in accurate separation of tumor from control samples. Thus, separation of tissue samples as in our initial proteome signature could be confirmed even with a different proteome analysis platform. In addition, detailed protein structure investigations enabled refining our proteome signature for invasive ductal breast carcinoma, opening the way to structure-/function studies with respect to disease processes and/or therapeutic intervention.

Mass spectrometric and peptide chip characterization of an assembled epitope: analysis of a polyclonal antibody model serum directed against the Sjøgren/systemic lupus erythematosus autoantigen TRIM21

“…For peptide mapping, 400 shots were acquired in positive reflector mode over the mass range of m / z 680–3500, and for high mass range measurements ( m / z 4000–20,000), 1000 shots were accumulated. Resulting mass spectra were processed using the FlexAnalysis 2.4 and BioTools 3.0 programs (Bruker Daltonik, Bremen, Germany) …”

Differential Proteomics of the Cerebral Cortex of Juvenile, Adult and Aged Rats: An Ontogenetic Study

“…Only those spots were considered to be significantly up-or downregulated that showed a SVQ (Spot Volume Quotient) of 0.6 or less and 1.67 or greater [26,31,34]. The differences were evaluated significantly if differentially expressed spots were detected in at least four gel images (correlated spots) belonging to one test group.…”

Mass spectrometric peptide analysis of 2DE‐separated mouse spinal cord and rat hippocampus proteins suggests an NGxG motif of importance for in vivo deamidation

“…B, C). The isoelectric heterogeneity of glyceraldehyde‐3‐phosphate dehydrogenase and other glycolytic enzymes is known and partial deamidations at NG sequences have been described in 2D gel‐separated spots of glyceraldehyde‐3‐phosphate dehydrogenase . Furthermore, a detailed analysis of post‐translational modifications using 2D gel‐separated human G3P revealed deamidations at various asparaginyl residues including some whose carboxyl side neighbor was not glycine, besides a multitude of further PTMs .…”