PTM Discovery Method for Automated Identification and Sequencing of Phosphopeptides Using the Q TRAP LC/MS/MS System

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Application Note LC/MS PTM Discovery Method for Automated Identification and Sequencing of Phosphopeptides Using the Q TRAP LC/MS/MS System Purpose This application note describes an automated

1 Application Note LC/MS PTM Discovery Method for Automated Identification and Sequencing of Phosphopeptides Using the Q TRAP LC/MS/MS System Purpose This application note describes an automated workflow to identify posttranslational modifications (PTMs) in proteins as well as determine the site of modification. The Q TRAP LC/MS/MS system with BioAnalyst and Pro ID software provide a single platform solution for automated PTM analysis. Overview The ultimate goal of protein discovery work is to identify and quantify proteins that are relevant to a given biological state, characterize those proteins with respect to post-translational modifications (PTMs) and unearth their networks of interactions in an effort to understand that biological state at the molecular level. The phosphorylation of proteins is one of the most studied of these PTMs because it is now thought that in a typical mammalian cell, as many as one-third of proteins are phosphorylated. Reversible protein phosphorylation mediates most of the signal transduction pathways in eukaryotic cells, controls cell processes such as metabolism and transcription, and plays a role in intercellular communication. Disruption of these phosphorylation cascades through mutation or misregulation play causal roles in many diseases. Q TRAP LC/MS/MS System A common way of studying phosphorylation has been through 2D gel electrophoresis on 32 P-labeled phosphopeptides the labeled spots are excised and analyzed by Edman sequencing, amino acid analysis, or mass spectrometry. In an effort to find an alternative to radio-labeling, more recent investigations have involved different MS techniques in combination to identify and characterize phosphorylated peptides. Single ion monitoring (SIM) or precursor ion (PI) scanning on a triple quadrupole mass spectrometer has been used to selectively determine which peptides contain a phosphate moiety. This is followed up with a second MS/MS step on either a quadrupole-tof instrument or an ion trap to obtain high sensitivity product ion spectra for sequence information. In practice, however, this is very challenging due to the very low concentration of phosphopeptides in highly complex protease digest mixtures. In addition, signals of phosphopeptides in the MS spectra are, in most cases, suppressed by the relatively high concentration of non-phosphopeptides present in proteolytic digests of these samples, which ionize much more efficiently. A better approach to selectively identify and characterize phosphopeptides from

2 complex digest mixtures is now available with the PTM discovery method using the Q TRAP LC/MS/MS system. The direct combination of triple quadrupole and ion trapping capabilities in the Q TRAP system presents new opportunities for the automated investigation of post-translational modifications in biological samples, including phosphorylation. For the first time, automated Information Dependent Acquisition (IDA) software (the acquisition feature that allows for MS/MS data to be obtained on the fly during LC/MS/MS) combines the unique specificity of precursor ion (PI) and neutral loss (NL) scans for PTM identification with high sensitivity ion trap MS/MS scans on an LC time scale. In addition, the ability to switch rapidly between positive and negative ion detection while performing these scans provides unique capabilities and maximum flexibility for automated PTM discovery. To identify the PTMs after IDA analysis, BioAnalyst and Pro ID software are used to determine the protein of origin for each modified peptide and to identify the site(s) of modification on the peptides. Key Features Single experiment identification and site determination of PTMs Q TRAP LC/MS/MS hybrid quadrupole linear ion trap combines highly selective precursor ion scans with high sensitivity MS/MS scans Information Dependent Acquisition (IDA) automatically links scan functions and derives maximum information from each run 7.3e7 7.0e7 6.5e7 6.0e7 5.5e7 5.0e7 4.5e7 4.0e7 3.5e7 3.0e7 2.5e7 2.0e7 1.5e7 1.0e7 0.0e Time, min Figure 1. Results of a looped experiment compare a full scan enhanced MS (EMS) scan that is continuously followed by a precursor ion (PI) scan during LC/MS. The blue trace is the total ion chromatogram for the full scan data, and the red trace is the total ion chromatogram for the PI scan, indicating the specific detection of the phosphopeptides during the LC/MS experiment. Advanced BioAnalyst software with Pro ID application identifies peptide sequence and site of PTM, and compares to theoretical ions Experimental Conditions Bovine alpha-2-hs-glycoprotein (fetuin) was purchased from Sigma. A soluble fraction of a yeast lysate obtained internally was also tested. Both samples were reduced and alkylated with iodoacetamide prior to analysis. 500 fmol of fetuin as well as 20 ug of soluble yeast lysate were loaded onto a one dimensional gel and separated. Bands from the 1D gel for fetuin and a high molecular weight protein from the yeast lysate were excised and digested out of the gel with trypsin. Both samples were analyzed using nanoflow chromatography coupled to a Q TRAP hybrid quadrupole linear ion trap LC/MS/MS system equipped with a Flow NanoSpray source. The protein digests extracted from the gel were separated using an Agilent 1100 Series Nanoflow LC System consisting of a nanoflow pump and a micro well-plate autosampler. A Valco switching valve, under Analyst software control, allowed online sample concentration on a C18 Cap Trap (Michrom Bioresources, Inc.). Separation was achieved on an LC Packings PepMap C18 column (75 µm x 15 cm), and a mobile phase of ACN/0.1% formic acid, with a 30 minute gradient elution and a flow of µl/min. For the selective identification of phosphorylation sites, the Q TRAP system was set to detect the characteristic PO 3 - fragment ion using a true precursor ion scan of a triple quadrupole at m/z 79 in negative ion mode.

4.0e8 3.5e8 3.0e8 2.5e8 2.0e8 1.5e8 1.0e8 5.0e7 0.0 1.8e4 1.6e4 1.4e4 1.2e4 1.0e4 8000.0 6000.0 4000.0 2000.0 1.9e6 1.8e6 1.8e8 6.

search or de novo sequencing to identify phosphorylation site Figure 2. Information Dependent Acquisition (IDA) links scan functions to fully automate the PTM discovery workflow.

3 4.0e8 3.5e8 3.0e8 2.5e8 2.0e8 1.5e8 1.0e8 5.0e e4 1.6e4 1.4e4 1.2e4 1.0e e6 1.8e6 1.8e8 6.0e5 negative ion mode precursor ion (PI) scan for m/z 79 as a survey scan switch polarities (700 msec) positive ion enhanced resolution (ER) scan positive ion enhanced product ion (EPI) scan database search or de novo sequencing to identify phosphorylation site Figure 2. Information Dependent Acquisition (IDA) links scan functions to fully automate the PTM discovery workflow. Precursor Ion Scan TIC of IDA run Negative Ion Precursor Ion Scan m/z M-2H Time, min e6 1.8e6 1.6e6 -/+ switching -/+ switching 700 msec e6 M+2H Figure 3. Automated analysis of phosphorylated peptides in tryptic digest. In the experiment shown, 500 fmol of the glycoprotein fetuin (less than 10% phosphorylated), was in-gel digested with trypsin and then run by nanoflow LC/MS/MS using IDA Positive Ion Enhanced Resolution Scan Enhanced Product Ion Scan MS/MS Results and Discussion First, to demonstrate the power of PI scanning to identify low level phosphorylated peptides in complex mixtures, the fetuin tryptic digest was analyzed by LC/MS on the Q TRAP system. For this experiment, the instrument was set to perform a full m/z range enhanced MS (EMS) scan followed directly by a 700 msec polarity switch and a PI scan for m/z 79, which is the mass of the phosphate ion (PO 3 - ) released from phosphopeptides. The results of this experiment are shown in Figure 1. Clearly, the PI scan allows selective identification of the phosphopeptides present in the complex tryptic digest of fetuin. For the PTM discovery experiment, the PI scan is combined with enhanced resolution (ER) and enhanced product ion (EPI or MS/MS) scans to obtain the sequence and identification site of the phosphorylation on the phosphopeptides. The method is automated by using IDA to seamlessly link the scan functions and gain the most information from any given run (Figure 2). In the automated PTM workflow, when an ion is detected in the PI scan, the instrument switches from negative to positive ion mode and performs an ER scan to determine the accurate mass and charge state of the peptide. This is followed by an MS/MS scan using the high sensitivity of the linear ion trap mode of the Q TRAP system. Such measurement cycles take less than five seconds to execute, rendering this approach feasible for HPLC analysis of complex mixtures even if more than one phosphopeptide elutes at the same time. An example of this is shown in Figure 3 for the glycoprotein fetuin, which was digested out of a onedimensional gel with trypsin and analyzed on the Q TRAP system.

software. The bold and yellow highlighted masses indicate the matching ions.

4 The resulting MS/MS data is then searched with Pro ID software to identify the peptide sequence and the site of phosphorylation. Figure 4 shows the detail of how the theoretical ions for the fetuin phosphopeptide sequence (fetuin residues ) HTFSGVASVESpSSGEAF match to the actual ions in the MS/MS spectrum in BioAnalyst software. The bold and yellow highlighted masses indicate the matching ions. The specific b-ions that match to the spectrum eliminate the possibility of phosphorylation at the amino acids in the N-terminal portion of the peptide, including the Thr at position 296 and Ser at positions 298 and 302 in the peptide sequence. In addition, the y-ions indicate the presence of the phosphorylation site at Ser-306 in the peptide. Although several phosphorylation sites on human and rat fetuin have been determined, phosphorylation on bovine fetuin is less well characterized. The phosphopeptide identified above has little response to positive mode ionization, and would not likely be selected as an MS ion for subsequent MS/MS analysis during IDA, making it very difficult to identify and sequence by current MS methods. In this automated PTM workflow, the HTFSGVASVESpSSGEAF peptide was easily detected by the negative ion PI scan, and subsequently sequenced by MS/MS leading to identification of the peptide and site of phosphorylation. Identification of phosphoproteins from complex samples As another example, 20 µg of a total yeast lysate was separated on a onedimensional gel, stained with a phosphopeptide specific stain PRO-Q Diamond phosphoprotein gel stain (Molecular Probes, Inc.) then the individual gel bands that stained positive for phosphorylation were cut manually from the gel. The proteins were digested with trypsin, and the peptides were extracted out of the gel slice. The PTM discovery method specific for phosphorylation was then employed to analyze these samples. Figure 5 shows an example of one of the high molecular weight yeast proteins identified as a phosphoprotein and the phosphorylation site that was identified using this method. Database searching of the MS/MS spectrum with the Pro ID software identified this protein as acetyl CoA synthetase, with a phosphorylation site at Ser e6 1.8e b6 1.5e6 1.3e6 b e6 9.0e5 7.0e5 6.0e5 5.0e5 3.0e5 1.0e5 b b y2 y b b b b b13 b Figure 4. MS/MS spectrum obtained from automated PTM discovery analysis of phosphorylated peptides in a fetuin tryptic digest. Pro ID software database searching identifies this peptide as being residues of fetuin, and the phosphorylation site is identified as Ser-306. The bold and yellow masses in the table above are the theoretical ions that are found in the spectrum (J=phosphoserine as defined in the Data Dictionary in BioAnalyst software).

5 Conclusions The automated PTM discovery method, using a precursor ion scan of -79 in negative ion mode, followed by an enhanced resolution scan and an enhanced product ion scan in positive mode, is a very effective tool for identifying phosphorylation sites in phosphoproteins in a single run. The high sensitivity of MS/MS data obtained using the linear ion trap features of the Q TRAP system allow for identification and sequencing of low level phosphopeptides. Using this method, a novel phosphorylation site was identified and sequenced on a commonly analyzed glycoprotein, fetuin, eluted from a one-dimensional gel. In addition, a novel phosphorylation site was identified and sequenced from a large unknown protein, acetyl CoA synthetase, which was extracted from a one-dimensional gel separation of a total yeast cell lysate. No other MS system on the market today has these combined features for protein identification and characterization of PTMs. Rel. Int. (%) 57% 50% 40% 30% 20% 10% 0% Precursor Ion Scan TIC of IDA run Time, min e5 Negative Ion Precursor Ion Scan m/z M-2H y b7-h3po4 b2-h3po4 b6-h3po b3-h3po4 y b5y5 b y y7 1.00e6 8.00e5 -/+ switching 6.00e5 700 msec4.00e5 700 msec 2.00e y y b b Positive Ion Enhanced Resolution Scan M+2H 2+ y y y Enhanced Product Ion Scan MS/MS y13 y Figure 5. Automated analysis of phosphorylated peptides from in-gel tryptic digest of high molecular weight yeast protein. In the experiment shown, the unknown yeast protein was digested with trypsin and then run by nanoflow LC/MS/MS using the PTM Discovery IDA method. From the spectrum, the peptide was identified as AVpSVSDLSYVANSQSSPLR from the yeast protein acetyl CoA synthetase, with a phosphorylation site at Ser This protein is known to be regulated by phosphorylation, but with no actual sites previously identified.

6 iscience. To better understand the complex interaction of biological systems, life scientists are developing revolutionary approaches to discovery that unite technology, informatics, and traditional laboratory research. In partnership with our customers, Applied Biosystems provides the innovative products, services, and knowledge resources that make this new, Integrated Science possible. Worldwide Sales Offices Applied Biosystems vast distribution and service network, composed of highly trained support and applications personnel, reaches 150 countries on six continents. For international office locations, please call the division headquarters or refer to our Web site at. Applera is committed to providing the world s leading technology and information for life scientists. Applera Corporation consists of the Applied Biosystems and Celera Genomics businesses. Applied Biosystems/MDS SCIEX is a joint venture between Applera Corporation and MDS, Inc. Headquarters 850 Lincoln Centre Drive Foster City, CA USA Phone: Toll Free: Fax: For Research Use Only. Not for use in diagnostic procedures. Applied Biosystems and Analyst are registered trademarks and AB (Design), Applera, iscience, iscience (Design), BioAnalyst, and NanoSpray are trademarks of Applera Corporation or its subsidiaries in the US and/or certain other countries. Q TRAP is a trademark of Applied Biosystems/MDS SCIEX. MDS and SCIEX are trademarks of MDS Inc. PRO-Q is a registered trademark of Molecular Probes, Inc Applied Biosystems. All rights reserved. Information subject to change without notice. Printed in the USA, 09/2003, LD Publication 114AP15-01

Multiplex Protein Quantitation using itraq Reagents in a Gel-Based Workflow

Multiplex Protein Quantitation using itraq Reagents in a Gel-Based Workflow Purpose Described herein is a workflow that combines the isobaric tagging reagents, itraq Reagents, with the separation power