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.

Affinity depletion of plasma and serum for mass spectrometry-based proteome analysis.

Protein biomarker discovery in blood plasma and serum is severely hampered by the vast dynamic range of the proteome. With protein concentrations spanning 12 orders of magnitude, conventional mass spectrometric analysis allows for detection of only a few low-abundance proteins. Prior depletion of high-abundant proteins from the sample can increase analytical depth considerably and has become a widely used practice. We describe in detail an affinity depletion method that selectively removes 14 of the most abundant proteins in plasma and serum.

Protein phosphorylation patterns in serum from schizophrenia patients and healthy controls.

Most proteomic studies to date have attempted to identify changes in protein levels without considering the effects of post-translational modifications (PTM). However, characteristic changes of PTM such as phosphorylation could be biologically informative, as these can give insights into disease and drug mechanisms of action at the functional level. With this in mind, we have conducted a comparative proteomic and phosphoproteomic analysis of blood sera from 20 antipsychotic-naïve schizophrenia patients and 20 matched healthy controls. We used immobilised metal ion affinity chromatography (IMAC) for enrichment of phosphoproteins combined with label-free liquid chromatography-mass spectrometry (LC-MS(E)) for identification and measurement of protein and phosphoprotein levels. The LC-MS(E) analysis of both IMAC-fractions resulted in identification of 35 proteins with altered levels in schizophrenia. Analysis of the enriched fraction resulted in identification of 72 phosphoproteins with altered phosphorylation patterns. Of these, 59 showed changes in phosphorylation only, with no overall change in protein levels. This study provided evidence that schizophrenia patients feature serum abnormalities in phosphorylation of proteins involved in acute phase response and coagulation pathways. Further studies of such phosphorylation-specific changes could lead to a better understanding of the molecular aetiology of schizophrenia, and provide a means of biomarker identification for clinical studies. This article is part of a Special Issue entitled: Integrated omics.

Clinical use of phosphorylated proteins in blood serum analysed by immobilised metal ion affinity chromatography and mass spectrometry.

The process of protein phosphorylation in cells is well studied in the context of a wide range of biologic functions such as signalling, cell cycle, cell growth and differentiation, and others. In contrast, little progress has been made in the investigation of protein phosphorylation specifically in blood. Here, we focussed on the phosphoproteome in human blood serum to study its extent and characteristics, and to explore the potential clinical utility. Immobilised metal ion affinity chromatography (IMAC) for the enrichment of intact phosphorylated proteins and label-free liquid chromatography-mass spectrometry (LC-MS(E)) were used for the molecular analysis of a large number of serum samples. To obtain high-confidence results, phosphorylated peptides had to be detected in at least 2 out of 3 technical replicates per sample and in >70% of the serum samples drawn from 80 volunteers. Individual analysis of these 80 non-pooled samples resulted in the detection of 5825 unique phosphorylated peptides after filtering, which corresponded to 502 unique proteins. The results provided evidence that blood serum may be an untapped source of phosphoproteins suitable for potential use in understanding disease pathophysiology and for identification of disease and drug response biomarkers. This article is part of a Special Issue entitled: Integrated omics.

Multidimensional protein fractionation of blood proteins coupled to data-independent nanoLC-MS/MS analysis.

In order to exploit human blood as a source of protein disease biomarkers, robust analytical methods are needed to overcome the inherent molecular complexity of this bio-fluid. We present the coupling of label-free SAX chromatography and IMAC to a data-independent nanoLC-MS/MS (nanoLC-MS(E)) platform for analysis of blood plasma and serum proteins. The methods were evaluated using protein standards added at different concentrations to two groups of samples. The results demonstrate that both techniques enable accurate protein quantitation using low sample volumes and a minimal number of fractions. Combining both methods, 883 unique proteins were identified, of which 423 proteins showed high reproducibility. The two approaches resulted in identification of unique molecular signatures with an overlap of approximately 30%, thus providing complimentary information on sub-proteomes. These methods are potentially useful for systems biology, biomarker discovery, and investigation of phosphoproteins in blood.