Characterization of the human serum depletome by label-free shotgun proteomics.

While it is known that immunoaffinity depletion of abundant proteins in serum removes additional proteins beyond those targeted, there has been little characterization of the co-depleted proteins in the high abundant fraction, which we refer to here as the "depletome". We present evidence of co-depletion of non-targeted proteins in human serum using a top-20 immunodepletion column, as shown by label-free liquid chromatography mass spectrometry (LC-MS(E)) profiling. This led to identification of 147 proteins which were specific for this fraction and comprised proteins with functions predominantly in binding and transport of nucleotides, metal ions, carbohydrates and lipids. These results suggest that further studies on this commonly ignored serum fraction may provide new insights into clinical proteomics.

Metabolic, hormonal and stress-related molecular changes in post-mortem pituitary glands from schizophrenia subjects.

OBJECTIVES: To identify a molecular profile for schizophrenia using post-mortem pituitaries from schizophrenia and control subjects. METHODS: Molecular profiling analysis of pituitaries from schizophrenia (n = 14) and control (n = 15) subjects was carried out using a combination of liquid chromatography tandem mass spectrometry (LC-MS(E)), multiplex analyte profiling (MAP), two-dimensional difference gel electrophoresis (2D-DIGE) and Western blot analysis. RESULTS: This led to identification of differentially expressed molecules in schizophrenia patients including hypothalamic-pituitary-adrenal axis-associated constituents such as cortisol, pro-adrenocorticotropic hormone, arginine vasopressin precursor, agouti-related protein, growth hormone, prolactin and secretagogin, as well as molecules associated with lipid transport and metabolism such as apolipoproteins A1, A2, C3 and H. Altered levels of secretagogin in serum from a cohort of living first onset schizophrenia patients were also detected, suggesting disease association and illustrating the potential for translating some components of this molecular profile to serum-based assays. CONCLUSIONS: Future studies on the molecules identified here may lead to new insights into schizophrenia pathophysiology and pave the way for translation of novel diagnostics for use in a clinical setting.

Identification of protein biomarkers in human serum using iTRAQ and shotgun mass spectrometry.

Blood serum is one of the easiest accessible sources of biomarkers and its proteome presents a significant parcel of immune system proteins. These proteins can provide not only biological explanation but also diagnostic and drug response answers independently of the type of disease or condition in question. Shotgun mass spectrometry has profoundly contributed to proteome analysis and is presently considered as an indispensible tool in the field of biomarker discovery. In addition, the multiplexing potential of isotopic labeling techniques such as iTRAQ can increase statistical relevance and accuracy of proteomic data through the simultaneous analysis of different biological samples. Here, we describe a complete protocol using iTRAQ in a shotgun proteomics workflow along with data analysis steps, customized for the challenges associated with the serum proteome.