Novel Relative ICPL Based Quantitative Phospho- and Glycoproteome Analysis Method

Abstract

Large scale proteomic analysis remains challenging partially because proteins are inhomogeneous and often influenced by a variety of structural modifications. In particular, these specific chemical modifications called posttranslational modifications (PTM) are crucial determinants for the protein function and biological role. Up to now there have been a growing number of studies describing the enrichment and identification of PTM. However, a significant dearth of data offering a reliable methodology for PTM quantification does exist. The present work aims at developing a label based protein PTM quantification strategy and demonstrating its value on comparative analysis of cells originating from two distinct prostate metastasis sites. PC3 and LNCaP cells isolated from bone and lymph node prostate cancer metastasis sites respectively, were lysed and spiked with three non-human proteins serving as internal standards. Following this, the samples were reduced and alkylated, digested with trypsin and subjected to peptide ICPL (isotope coded protein label) labeling. The two peptide containing samples were joined together followed by the affinity isolation of phospho- (using TiO2 metal affinity chromatography) and glycopeptides (oxidized glycans were bound on hydrazide resin). The enriched fraction as well as the flow-through were analyzed on a 2D-(SCX and C18-RP)-nano-HPLC system. The peptide identification and quantification was conducted using electrospray ion-trap mass spectrometer (Bruker, HCT-ultra). Validation of the differentially modulated proteins was conducted in several biological and technical replicates using the label free MSe based quantification strategy. This PTM based, novel relative protein quantification using post-digest ICPL has detected over 598 individual proteins. Of these more than 95 % have been successfully quantified. PTM enrichment methodologies allowed an isolation rate of 91 % and 50 % for phosphorylated and glycosylated proteins respectively. The detailed comparison of PC3 and LNCaP cells has shown specific overexpression of selected proteins indicating differences between these two prostate metastatic cell lines. Several of these modulated proteins have been previously described to be related to prostate cancer (e.g. annexin A2 and vimentin) while others could be considered as potentially novel. These proteins might be implicated in the fundamental process related to metastasis dissemination. However, because of the known discrepancy between cell systems and clinical material, the present study can be regarded only as a step towards elucidation of these complex interactions