Assay Development for the Determination of Phosphorylation Stoichiometry Using Multiple Reaction Monitoring Methods with and without Phosphatase Treatment: Application to Breast Cancer Signaling Pathways

Abstract

We have developed a phosphatase-based phosphopeptide quantitation (PPQ) method for determining phosphorylation stoichiometry in complex biological samples. This PPQ method is based on enzymatic dephosphorylation, combined with specific and accurate peptide identification and quantification by multiple reaction monitoring (MRM) detection with stable-isotope-labeled standard peptides. In contrast with the classical MRM methods for the quantitation of phosphorylation stoichiometry, the PPQ-MRM method needs only one non-phosphorylated SIS (stable isotope-coded standard) and two analyses (one for the untreated and one for the phosphatase-treated sample), from which the expression and modification levels can accurately be determined. From these analyses, the % phosphorylation can be determined. In this manuscript, we compare the PPQ-MRM method with an MRM method without phosphatase, and demonstrate the application of these methods to the detection and quantitation of phosphorylation of the classic phosphorylated breast cancer biomarkers (ERα and HER2), and for phosphorylated RAF and ERK1, which also contain phosphorylation sites with important biological implications. Using synthetic peptides spiked into a complex protein digest, we were able to use our PPQ-MRM method to accurately determine the total phosphorylation stoichiometry on specific peptides, as well as the absolute amount of the peptide and phosphopeptide present. Analyses of samples containing ERα protein revealed that the PPQ-MRM is capable of determining phosphorylation stoichiometry in proteins from cell lines, and is in good agreement with determinations obtained using the direct MRM approach in terms of phosphorylation and total protein amount.

Differential Mass Spectrometry Profiles of Tau Protein in the Cerebrospinal Fluid of Patients with Alzheimer’s Disease, Progressive Supranuclear Palsy, and Dementia with Lewy Bodies

“…The relative increase of pep 195-209 in AD, however, is very puzzling and could correspond also to differences in post-translational modification or in the amount of Tau fragments covering this sequence. Further studies, and in particular MS quantification of the corresponding phosphopeptides [36] would be needed to fully explain these observations.…”

Quantitative Targeted Absolute Proteomics-Based Adme Research as A New Path to Drug Discovery and Development: Methodology, Advantages, Strategy, and Prospects

“…Quantification of other phosphorylated proteins, such as focal adhesion kinase, estradiol receptor ", and HER2, by LC-MS/MS has also been reported. 20,21 Simultaneous quantification of multiple proteins is another advantage of LC-MS/MS-based quantification (Table 1). Thirty-seven proteins can currently be quantified simultaneously, and we have reported the simultaneous quantification of 34 transporters and two membrane proteins in mouse brain capillaries, liver, and kidney.…”

Liquid Chromatography and Mass Spectrometry (LC-MS) Based Strategies for Quantitative Phosphoproteomics

“…Because of two levels of mass selection and the unique operation mode with non-scan recorded for full MS, SRM-MS features high sensitivity, high selectivity and wide dynamic range (up to five orders of magnitude). [152][153][154][155]. As a result, SRM-MS has been increasingly applied to quantitative phosphoproteomics [152][153][154][155].…”

“…[152][153][154][155]. As a result, SRM-MS has been increasingly applied to quantitative phosphoproteomics [152][153][154][155]. In 2002, Ruse and coworkers developed a SRM-MS method to quantify site-specific protein phosphorylation, and demonstrated the time-dependent phosphorylation at a PKC-preferred site, Ser44 in the peptide 41 ISASPR 45 and the concomitant consumption of the unphosphorylated peptide through monitoring the time course of a protein kinase C beta II reaction.…”