1. Monitoring of temporal changes in phosphorylation states of proteins using mass spectrometry and a chemical labeling strategy RESULTS INTRODUCTION It is well known that protein phosphorylation is one of the most important post-translational modifications (PTMs). It plays a key role in signal transduction pathways and thus is of significant importance in disease- related studies. Protein phosphorylation is also known to be both spatial and dynamic in its distribution. Therefore, it is advantageous to develop methods for the identification and quantification of individual phosphorylation sites in different cell states. A complete workflow is necessary which provides information both on the location of the phosphorylation sites and their change in occupancy under varied conditions. However existing non-mass spectrometric techniques to study phosphorylation, such as Western blotting or a radio-labeling, lack the ability to decipher the independent changes in phosphorylation at individual sites. Presented here is a quantitative approach for monitoring changes at individual phosphorylation sites using iTRAQ™ reagents and the 4000 Q TRAP ® system. To establish this quantitative workflow and develop a phosphorylation assay, a model system was chosen involving the kinase JNK1 and the substrate protein Myelin Basic Protein (MBP). JNK1, a member of the MAP kinase family, specifically phosphorylates Serine and Threonine residues which are N-terminal to a Proline residue. The time dependent analysis of the phosphorylation of MBP by JNK1 was performed in vitro as described in Figure 1. MATERIALS AND METHODS Preparation of Samples: JNK1 kinase was provided by Carna Bioscience Inc. MBP (Myelin basic protein) was obtained from Upstate. A time course of phosphorylation of MBP was created by incubating it with the JNK1 kinase for 0, 5, 30 and 60 minutes. Protein samples were digested with trypsin and labeled with iTRAQ™ reagents according to the Applied Biosystems protocol. Chromatography: The iTRAQ™ reagent labeled sample was analyzed by nano LC-MS/MS using a C18 column (3  m,100  m x 5 cm, KYA Technologies Corporation) with a 0-28% acetonitrile over 45 mins on a DiNa nano LC system (KYA Tech. Co). Mass Spectrometry: Information Dependent Analysis (IDA) analysis and Multiple Reaction Monitoring (MRM) were performed using the NanoSpray ® source on a 4000 Q TRAP ® system. Data Processing: Identification and relative quantification analysis was performed using the Paragon™ Database Search Algorithm and Pro Group™ Algorithm in ProteinPilot™ Software. MIDAS™ workflow: MRM channels of Phosphopeptides were built using the MS/MS data in previous IDA experiment. Fumihiko Tsuchiya 1, Christie Hunter 2, Mamoru Matsubara 3, ： 1 Applied Biosystems, Tokyo, Japan, 2 Applied Biosystems, Foster City, USA, 3 Kyoto Gakuen University, Kyoto, Japan, Poster 85-S Myelin Basic Protein (MBP) was easily identified with ProteinPilot ™ software with 80% sequence coverage (Figure 2). All peptides containing the consensus motif of a MAP kinase (.. (S/T)P) were detected in a single run. Figure 2. Identification of Myelin Basic Protein (MBP). High quality chromatography (top) yielded many quality MS/MS spectra with total protein sequence coverage of 80% (bottom). Figure 3. Classification of Phosphorylation Types. Classification of Phosphorylated Peptides The iTRAQ™ reagent reporter ion patterns can be used to classify the identified peptides into groups (Figure 3). The time course of phosphorylation at individual phosphorylation sites of the range can be understood by analyzing the iTRAQ™ reagent reporter ions between m/z 114 and 117 in the MS/MS spectra. Peptides that are not phosphorylated, or where the phosphorylation levels does not change throughout the time course, are Type C or constant (Figure 3, left); their iTRAQ™ reagent reporter ions are all or equal intensity (Figure 4). A peptide that shows increased occupancy of a phosphorylation site in the presence of the kinase is Type I, where the intensity of each successive reporter ion increases over time (Figure 3, middle). This peptide which possesses a phosphorylation site can also be observed as a decrease in the amount of its non-phosphorylated counterpart (Figure 3, right). Peptides with no phosphorylation sites (Type C) will show constant signal in the iTRAQ™ reagent reporter ions during the time course. Peptides containing phosphorylation sites will have complementary patterns, the phosphorylated peptide signals will increase during the time course (Type I) while the unphosphorylated peptide signal will decrease during the time course (Type D). Figure 1. Experimental Design. JNK1 knase was incubated with the model substrate MBP. Aliquots were taken over time and tryptically digested and labeled with iTRAQ™ reagent. MS/MS spectra were used to identify peptides and the sites of phosphorylation and the iTRAQ reagent reporter ions provided the relative quantification information between each time point. High quality MS/MS spectra allow for the simultaneous identification and quantification of individual peptides over time in this kinase assay. Shown in Figure 4 is an example of a peptide that does not get phosphorylated by the JNK1 kinase as seen by the pattern of the iTRAQ™ reagent reporter ions (a type C pattern). Peptides that are phosphorylated over time during the assay will have a pattern showing an increase in the iTRAQ™ reagent reporter ions (Type I). As an example, an MS/MS spectrum and the reporter ion regions of the relevant spectra for the phosphorylation of the NIVTPRTPPPSQGK peptide are shown in Figure 5. Phosphorylation of one or more of the adjacent threonine residues (Thr94 and Thr97) can cause a missed cleavage at the central arginine residue during tryptic digestion. This peptide becomes doubly phosphorylated on the two threonine residues during the kinase assay, showing a constant increase in the reporter ions for the doubly phosphorylated peptide. Both singly phosphorylated versions of this sequence are only observed transiently, appearing in the first 5 minutes, then disappearing (Figure 5). The two peptides corresponding to the completely unphosphorylated peptide decrease over time. Dynamic Changes in Site Specific Phosphorylation This peptide does not possess a phosphorylation site and therefore shows constant signal intensity of the iTRAQ™ reagent reporter ions across the four samples in the time course (Type C peptide). Figure 5. Quantifying the Changes in Phosphorylation Over Time of the Doubly Phosphorylated Peptide. High quality MS/MS spectra of the phosphorylated peptides enable unambiguous identification of the sites of modification, as illustrated by the spectrum of the singly phosphorylated NIVTPRpTPPPSQGK peptide (left). Examination of the iTRAQ™ reagent ions shows the amount of this peptide increasing during the first 5 minutes, then disappearing in subsequent time points (left, inset). In MS/MS spectra of related peptides, it can be observed that the singly phosphorylated peptide NIVpTPRTPPPSQGK also shows increasing levels initially, then decreases over time (right, top). This is accompanied by the increase over time of the doubly phosphorylated NIVpTPRpTPPPSQGK (right, top). At the same time, the amounts of the corresponding unphosphorylated peptides covering this same sequence decrease over time (right, bottom). MIDAS™ workflow is a key tool for developing MRM quantitation assays of the characterized phosphopeptides. Peptides (with and without phosphate modifications) can be directly monitored with MRMs using rapid reverse phase chromatography. Figure 6 shows the results of the MRM assay development for the three phosphopeptides that were determined to be phosphorylated by JNK1 in the iTRAQ reagent experiment. In a short 20 min gradient, full scan ion trap MS/MS was obtained for each main MRM peak, confirming the three phosphorylated with high quality MS/MS. A peptide SGpSPMAR was detected as broad but dominant early-eluting peak because it is very hydrophilic peptide. Specific MRMs can independently detect two peptide isoforms, NIVpTPRpTPPPSQGK and NIVTPRpTPPPSQGK, even though these two peptide peaks overlap. Figure 6. MRM initiated detection of three objective phosphopeptides and identification of phosphorylated sites using MIDAS™ Workflow. Shown are the high quality MS/MS spectra obtained from the MIDAS™ Workflow, reasonably complete sequence ladders (y / b ions and neutral loss from y / b ions) were obtained to confirm the identity of the peptides and the location of the phosphate modifications. Quantitation of Phosphorylation using MRMs and MIDAS ™ workflow Figure 4. Non-Phosphorylated Peptide. 1. Using the iTRAQ™ reagents and the 4000 Q TRAP ® system, the time course of site-specific phosphorylation on the Myelin Basic Protein by JNK1 kinase could be monitored. 2. The observed time courses could be classified into three different types, Constant (C), Increasing (I), and Decreasing (D). 3. A better understanding of the complexities of this system is obtained when the time-dependent effects are monitored. 4. Now that this method has been established, it will provide an excellent method for screening for kinase inhibitors. 5. MIDAS™ workflow improves the efficiency of MRM method development, to create targeted quantitative MRM assays for phosphorylation analysis. Applied Biosystems / MDS Sciex would like to thank Dr. Kenich Kudo for technical support of this project and for supplying the chromatography column used. Applied Biosystems, AB (design) and Applera are registered trademarks and iTRAQ is a trademark of Applera Corporation or its subsidiaries in the U.S. and or certain other countries. Paragon, Pro Group and ProteinPilot are trademarks and Q TRAP and NanoSpray are registered trademarks of Applied Biosystems/MDS SCIEX. MDS and SCIEX are registered trademarks of MIDS, Inc. All other trademarks are the sole property of their respective owners. © 2007 Applera Corporation and MDS, Inc. All rights reserved. Information subject to change without notice. For Research Use Only. Not for use in diagnostic procedures. CONCLUSIONS ACKNOWLEDGEMENTS / TRADEMARKS iTRAQ SGpSPMAR iTRAQ NIVpTPRpTPPPSQGK iTRAQ iTRAQ NIVTPRpTPPPSQGK iTRAQ Type C