In Situ Sample Processing Approach (iSPA) for Comprehensive Quantitative Phosphoproteome Analysis Junfeng Huang, †,‡ Hongqiang Qin, † Jing Dong, † Chunxia Song, † Yangyang Bian, †,‡ Mingming Dong, †,‡ Kai Cheng, †,‡ Fangjun Wang, † Deguang Sun, § Liming Wang, § Mingliang Ye,* ,† and Hanfa Zou* ,† † CAS Key Lab of Separation Sciences for Analytical Chemistry National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China ‡ University of Chinese Academy of Sciences, Beijing 100049, China § The Second Affiliated Hospital of Dalian Medical University, Dalian 116027, China * S Supporting Information ABSTRACT: Current sample preparation protocols for quantitative phosphopro- teome analysis are tedious and time-consuming. Here, a facile in situ sample processing approach (iSPA) is developed by using macroporous Ti(IV)-IMAC microspheres as the preparation “beds”, where all sample preparation procedures including the enrichment of phosphoproteins, tryptic digestion of proteins, enrichment, and isotope labeling of phosphopeptides are performed in situ sequentially. As a result of the in situ processing design and the seamless procedures, extra steps for desalting and buffer exchanging, which are always required in conventional approaches, are avoided, and the sample loss and contamination could be greatly reduced. Thus, better sensitivity and accuracy for the quantitative phosphoproteome analysis were obtained. This strategy was further applied to differential phosphoproteome analysis of human liver tissues with or without hepatocellular carcinoma (HCC). In total, 8548 phosphorylation sites were confidently quantified from three replicate analyses of 0.5 mg of human liver protein extracts. KEYWORDS: in situ sample processing approach, phosphoprotein enrichment, on-beads digestion, dimethyl labeling, solid phase labeling, phosphoproteome quantification, human liver, hepatocellular carcinoma ■ INTRODUCTION Protein phosphorylation, as one of the most important post- translational modifications (PTMs), plays important roles in regulating biological processes such as signal transduction and cell division, growth, differentiation and apoptosis. 1 About 30% of cellular proteins can be phosphorylated during the cell cycle, and abnormal protein phosphorylation events are always accompanied by many diseases, such as cancers, diabetes, chronic inflammation, and neurodegeneration. 2 Comprehensive identification and quantification of protein phosphorylation is helpful for understanding biological processes and for aiding disease diagnosis. 3-5 Compared with non-phosphoproteins, the abundance of phosphoproteins is much lower, and for bottom- up phosphoproteomics the detection of phosphopeptides is much less sensitive than for non-phosphopeptides due to the low ionization efficiency in MS. Therefore, direct identification of phosphopeptides in a proteome digest by LC-MS/MS analysis is almost impossible. Thus, selective enrichment of phosphoproteins or phosphopeptides before MS analysis is vital for the phosphorylation analysis. 6 Recently, there are many methods that have been developed for the phosphopeptides enrichment, such as strong cation/ anion exchange chromatography (SCX/SAX), immobilized metal ion and metal oxide affinity chromatography (IMAC and MOAC), etc. 7-10 Thousands of phosphopeptides could be routinely identified in one LC-MS/MS analysis using these methods. However, the sample preparation procedures of these enrichment methods are often performed in solution, and for the quantitative phosphoproteome analysis, an additional sample preparation step, i.e., stable isotope labeling, is required. Because of the incompatibility of buffers used in these different procedures including digestion, labeling, and phosphopeptide enrichment, buffer exchanges are always required. The multiple steps of desalting and solution transfer processes may lead to serious sample loss and contamination, which will compromise the performance for phosphoproteomics analysis. Additionally, because of the tedious sample preparation processes, the conventional approach is very time-consuming and typically costs over 2 days for the sample preparation. 6 Moreover, because of the complexities of mammalian tissue proteomes and their low phosphorylation level, the proteome-wide identification and quantification of protein phosphorylation from mammalian tissues is still a challenge. 3,11 Thus, new strategies with optimized sample preparation procedures and reduced preparation time to improve the Received: January 25, 2014 Published: July 29, 2014 Article pubs.acs.org/jpr © 2014 American Chemical Society 3896 dx.doi.org/10.1021/pr500454g | J. Proteome Res. 2014, 13, 3896-3904