Laser-Induced Dissociation of Phosphorylated Peptides Using
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09_Cotter 10/19/07 7:57 AM Page 133 Clinical Proteomics Copyright © 2006 Humana Press Inc. All rights of any nature whatsoever are reserved. ISSN 1542-6416/06/02:133–144/$30.00 (Online) Original Article Laser-Induced Dissociation of Phosphorylated Peptides Using Matrix Assisted Laser Desorption/Ionization Tandem Time-of-Flight Mass Spectrometry Dongxia Wang,1 Philip A. Cole,2 and Robert J. Cotter2,* 1Biotechnology Core Facility, National Center for Infectious Disease, Center for Disease Control and Prevention, Atlanta, GA; 2Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD site or concensus sequence is present in a given Abstract tryptic peptide. Here, we investigated the frag- Reversible phosphorylation is one of the mentation of phosphorylated peptides under most important posttranslational modifications laser-induced dissociation (LID) using a of cellular proteins. Mass spectrometry is a MALDI-time-of-flight mass spectrometer with a widely used technique in the characterization of curved-field reflectron. Our data demonstrated phosphorylated proteins and peptides. Similar that intact fragments bearing phosphorylated to nonmodified peptides, sequence information residues were produced from all tested peptides for phosphopeptides digested from proteins can that contain at least one and up to four be obtained by tandem mass analysis using phosphorylation sites at serine, threonine, or either electrospray ionization or matrix assisted tyrosine residues. In addition, the LID of laser desorption/ionization (MALDI) mass phosphopeptides derivatized by N-terminal spectrometry. However, the facile loss of neutral sulfonation yields simplified MS/MS spectra, phosphoric acid (H3PO4) or HPO3 from precur- suggesting the combination of these two types sor ions and fragment ions hampers the precise of spectra could provide an effective approach determination of phosphorylation site, particu- to the characterization of proteins modified by larly if more than one potential phosphorylation phosphorylation. *Author to whom all correspondence and reprint requests should be addressed: Robert J. Cotter, Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD. E-mail: [email protected]. 133 09_Cotter 10/19/07 7:57 AM Page 134 134 ______________________________________________________________________ Wang, Cole, and Cotter Key Words: Phosphorylation; MALDI; time-of- expense of other pathways that would produce flight mass spectrometry; curved-field reflectron; sequence ions; particularly those that retain the N-terminal sulfonation; fragmentation. phosphate group, so that it is not possible to fix Introduction the actual position of the phosphate on the pep- tide sequence. In tandem instruments which uti- Reversible phosphorylation is one of the lize low energy CID, that is: quadrupole ion most common and important regulatory mod- traps, hybrid quadrupole/time-of-flight mass ifications of cellular proteins (1). This modifi- spectrometers, Fourier transform mass spec- cation plays a crucial role in maintaining and trometers, and so on, ions are initially cooled regulating protein structure and function in a through collisions with inert gas in an RF ion number of biological processes and signal guide, retaining little of the internal energy transduction pathways. Among 6000 yeast pro- derived from the ionization process as they teins recently reported in the genomic database enter the mass analyzer. Following precursor of budding yeast Saccharomyces cerevisiae, about ion selection multiple low-energy collisions 250 are protein kinases that are responsible for increase the internal energy slowly, favoring substrate phosphorylation (2). Phosphorylation fragmentation of the weaker bonds that leads usually occurs on one of three amino acid not only to the facile loss of anionic groups residues: serine, threonine, or tyrosine. The (such as phosphate) and other posttranslational majority of phosphorylation occurs on serine modifications, but also to preferential cleavages residue (~90%), with only a small number of at proline, aspartic acid, and glutamic acid phosphoproteins containing phosphothreonine residues (8,9). (~10%) and phosphotyrosine (~1%). The iden- Electron capture dissociation was introduced tification and characterization of phosphory- recently as a fragmentation method for Fourier lated proteins is a major effort in proteomics. transform mass spectrometers (10–12) and has Mass spectrometry has become a powerful shown considerable promise as an activation tool for protein identification, providing rapid method for elucidating phosphorylation sites and reliable peptide sequencing using a number (13–15). For peptides (and for proteins in so- of fragmentation methods. The determination of called top down methods [16]) electron capture phosphorylation sites within modified proteins, dissociation produces predominant c and z. (or however, remains a challenge. This is primarily c. and z) fragment ions resulting from the cleav- because the most common method used to age of the amine backbone bond of precursor induce fragmentation in electrospray ionization ions. Fragmentation is more distributed, rather (ESI) and matrix assisted laser desorption/ion- than selective for specific residues or side ization (MALDI) mass spectrometers, low- groups, and, therefore, an abundance of energy collision-induced dissociation (CID), sequence-specific fragment ions are available results in significant loss of neutral phosphoric for the determination of both sequences and acid (H3PO4 98 Da) or HPO3 (80 Da) from phos- the location of posttranslational modifications. phorylated amino acid residues (3–7). The facile Similarly, it has also been suggested that loss of these groups could result from either MALDI mass spectra obtained on time-of-flight gas-phase β-elimination of the phosphate ester mass spectrometers produce charge remote frag- from phosphoserine or phosphothreonine mentation, both from post-source decay and CID residues, or from a two-step process involv- processes (17). Similar to the high-energy CID ing the loss of HPO3 followed by the loss of processes described previously for sector mass H2O from phosphotyrosine residues. This spectrometers (18), fragmentation is not local- fragmentation pathway is generally at the ized at specific sites, but well-distributed and Clinical Proteomics ________________________________________________________________ Volume 2, 2006 09_Cotter 10/19/07 7:57 AM Page 135 Laser-Induced Dissociation of Phosphorylated Peptides ____________________________________________ 135 therefore more informative of structure. The RELEELNVPGEIVEpSLpSpSEESITR, were pur- tandem time-of-flight (TOF/TOF) mass spec- chased from Sigma (St. Louis, MO). α-Cyano- trometer provides a particularly effective instru- 4-hydroxycinnamic acid was from Aldrich mental configuration for observing charge (Milwaukee, WI). The synthetic peptides, remote fragmentation of MALDI ions with better RLEpSR and RLEpTR, were prepared by the mass selection than the single analyzer instru- Synthesis and Sequencing Facility at the Johns ments for which post-source decay was initially Hopkins University School of Medicine (Balti- developed (19). Unlike most other tandem and more, MD). Peptides VSSDGHEpYIYVDP- hybrid instruments in which precursor ions are MQLPY and VSSDGHEpYIpYVDPMQLPY collisionally cooled, the internal energy result- were gifts from Dr. Akhilesh Pandey. All other ing from the initial MALDI processes can be peptides were synthesized with FMOC chem- used to drive the fragmentation. Such laser- istry by solid phase peptide synthesis and induced dissociation (LID) processes can also purified by reverse-phase high-performance be complemented by the inclusion of a collision liquid chromatography. chamber to produce single, high energy collisions that also lead to charge remote fragmen- Sulfonation tation. The motivation for this current work was Phosphopeptides were derivatized with the possibility that well-distributed fragmentation SPITC as previously described (21). In brief, could also be observed for phosphorylated pep- the reagent solution was prepared by dissolv- tides and could provide sequence-specific ing SPITC (10 mg/mL) in 20 mM NaHCO3 fragments that were more informative for deter- (pH ~ 9.0). The sulfonation reaction was car- mining phosphorylation sites. ried out in a 0.6-mL Eppendorf tube by mixing Here, we present our investigation of the 9 µL of reagent solution with 1 µL of peptide fragmentation of phosphorylated peptides by solution (approx 10–100 pmol). After incuba- LID using a MALDI-TOF/TOF mass spectrom- tion for 30 min at 55°C the reaction was ter- eter with a curved-field reflectron (20). Our minated by adding 1 µL of 1% trifluoracetic data demonstrate that intact fragments bearing acid (TFA). The sample was then loaded onto phosphorylated residues could be produced a micropipet tip (C18 OMIX, Varian, Lake from a series of peptides with from one to four Forest, CA), washed with 3 × 10 µL of 0.1% phosphorylation sites at serine, threonine, or TFA and followed by eluting with 10 µLof tyrosine residues; and that these could be used 75% acetonitrile/0.1% TFA. The solution was effectively to map the phosphorylation sites. In evaporated to dryness by SpeedVac and then µ addition, we utilized a method developed resuspended with 10 L of ddH2O. recently in our laboratory (21) to form N-termi- nal sulfonate derivatives of phosphopeptides,