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J Biomol Tech. 2007 February; 18(1): 65–66.
PMCID: PMC2291824

P189-T IMAC Enrichment of Phosphopeptides Coupled with 18O Labeling: Quantitative Phosphoproteomics

Abstract

The up- and down-regulation of protein expression has long been a focus of proteomics researchers and has given rise to several mass spectrometry–based quantitation techniques. In the past decade, a great deal of attention has turned toward the subproteomes, particularly those representing post-translational modifications (PTMs), such as phosphorylation. Those PTMs often play key roles in signaling cascades, and affect a number of cellular processes; their study offers insight into intracellular function. We illustrate here phosphoproteome quantitation through the pairing of immobilized metal-affinity chromatography (IMAC) and 18O labeling, which allows for the determination of modulated phosphorylated species in response to external stressors. Following application of varying biological stressors and isolation of each resultant proteome, the samples were proteolytically cleaved using immobilized trypsin. IMAC enrichment of phosphorylated peptides was accomplished using a nitrilotriacetic acid (NTA) analog bonded to silica, presenting Ga3+ as the chelated metal species. Immobilized trypsin was also employed for incorporation of 18O into the sample for quantitative global labeling. It was determined that IMAC enrichment utilizing this combination of chelate, substrate, and metal provided significant enrichment of phosphopeptides that afforded signal levels, which allowed for quantitative analysis of the phosphoproteome. The use of 18O labeling of the samples permitted a global approach to targeting the phosphoproteome concurrent with the non-phosphorylated component. Using MS analysis before and after IMAC-based phosphopeptide enrichment revealed a distinction in regulation of expression and modulation through phosphorylation of the proteins. The coupling of phosphopeptide enrichment with global isotopic labeling provides a valuable and enabling resource for the vastly under-explored area of quantitative phosphoproteomics.


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