Mechanistic Insights into the Multistage Gas-Phase Fragmentation Behavior of Phosphoserine- and Phosphothreonine-Containing Peptides

We have previously coupled stable isotope dimethyl labeling with IMAC enrichment for quantifying the extent of protein phosphorylation in vivo. The enhanced a 1 signal of dimethylated peptides served as a unique mass tag for unequivocal identification of the N-terminal amino acids. In this study, we demonstrate that the a 1 ion could further assist in mapping the precise phosphorylation site near the N-terminal region and allow the determination of the exact site and level of phosphorylation in one step by stable isotope dimethyl labeling. We show that the a 1 ion signal was suppressed for dimethylated peptides with a phosphorylation site at the N-terminus Ser/Thr residue (N-p*Ser/Thr) but was still enhanced for N-terminus Tyr residue (N-p*Tyr) or internal Ser/Thr residues (-p*Ser/Thr). Based on the dominant de-phosphorylated molecular ions and b-H 3 PO 4 ions for N-p*Ser/Thr, we propose that dimethyl labeling increases the basicity of the N-terminus and accelerates the dephosphorylation for N-p*Ser/Thr precursors, which, however, suppresses the a 1 ion enhancement due to the resulting unsaturated covalent bond on C ␣ of the N-terminus amino acid. Using this method, we excluded three Ser/Thr phosphorylation sites in A431 cells, two of which, however, were previously reported to be phosphorylation sites; we confirmed three known phosphorylation sites in A431 cells and quantified their ratios upon EGF treatment. Notably, we identified a novel phosphorylation site on Ser43 residue at N-terminus of the tryptic peptide derived from SVH protein in pregnant rat uteri. SVH protein has not been reported or implied with any phosphorylation event, and our data show that the Ser43 of SVH is an intrinsic phosphorylation site in pregnant rat uteri and that its phosphorylation level was slightly decreased upon c-AMP treatment. (J Am Soc Mass Spectrom 2010, 21, 460 -471)

Section: Fragmentation Pathwaysupporting

confidence: 93%

Section: Fragmentation Pathwaymentioning

confidence: 99%

Mapping N-terminus phosphorylation sites and quantitation by stable isotope dimethyl labeling

Hsu

Huang

et al. 2010

“…This mechanism has been shown to be inconsistent with newer labeling data and CID-MS 3 investigation of product ions. A more likely mechanism involves proton-induced elimination with neighboring participation of amino [56] or amide groups [57] from the N-terminal site of the phosphoserine residue, as recently reviewed [58]. Obviously, none of these mechanisms can apply to the elimination of phosphoric acid from deaminated serine residues in the z ions, which have vinylic α-hydrogen atoms and are missing the N-terminal functional groups.…”

Section: Mechanisms For Z-ion Dissociationsmentioning

confidence: 99%

Dipole-Guided Electron Capture Causes Abnormal Dissociations of Phosphorylated Pentapeptides

Moss

Chung

Wyer

et al. 2011

Electron transfer and capture mass spectra of a series of doubly charged ions that were phosphorylated pentapeptides of a tryptic type (pS,A,A,A,R) showed conspicuous differences in dissociations of charge-reduced ions. Electron transfer from both gaseous cesium atoms at 100 keV kinetic energies and fluoranthene anion radicals in an ion trap resulted in the loss of a hydrogen atom, ammonia, and backbone cleavages forming complete series of sequence z ions. Elimination of phosphoric acid was negligible. In contrast, capture of lowenergy electrons by doubly charged ions in a Penning ion trap induced loss of a hydrogen atom followed by elimination of phosphoric acid as the dominant dissociation channel. Backbone dissociations of charge-reduced ions also occurred but were accompanied by extensive fragmentation of the primary products. z-Ions that were terminated with a deaminated phosphoserine radical competitively eliminated phosphoric acid and H 2 PO 4 radicals. A mechanism is proposed for this novel dissociation on the basis of a computational analysis of reaction pathways and transition states. Electronic structure theory calculations in combination with extensive molecular dynamics mapping of the potential energy surface provided structures for the precursor phosphopeptide dications. Electron attachment produces a multitude of low lying electronic states in charge-reduced ions that determine their reactivity in backbone dissociations and H-atom loss. The predominant loss of H atoms in ECD is explained by a distortion of the Rydberg orbital space by the strong dipolar field of the peptide dication framework. The dipolar field steers the incoming electron to preferentially attach to the positively charged arginine side chain to form guanidinium radicals and trigger their dissociations.

“…Collectively, these results have revealed that peptides decorated with labile modifications often are not well behaved under low-energy MS/MS conditions. For example, facile loss of phosphoric acid from the side chains of serine-and threoninephosphorylated peptides often limits the available sequence-specific information contained in associated MS/MS spectra [17,18]. Interestingly, recent reports [19,20] suggest that higher energy, or "triple quadrupole like," MS/MS can provide improved sequence analysis, particularly for phosphorylated peptides.…”

mentioning

confidence: 99%

Optimized orbitrap HCD for quantitative analysis of phosphopeptides

Zhang

Ficarro

et al. 2009

118

Despite the tremendous commercial success of radio frequency quadrupole ion traps for bottom-up proteomics studies, there is growing evidence that peptides decorated with labile post-translational modifications are less amenable to low-energy, resonate excitation MS/MS analysis. Moreover, multiplexed stable isotope reagents designed for MS/MSbased quantification of peptides rely on accurate and robust detection of low-mass fragments for all precursors. Collectively these observations suggest that beam-type or tandem in-space MS/MS measurements, such as that available on traditional triple quadrupole mass spectrometers, may provide beneficial figures of merit for quantitative proteomics analyses. The recent introduction of a multipole collision cell adjacent to an Orbitrap mass analyzer provides for higher energy collisionally activated dissociation (HCD) with efficient capture of fragment ions over a wide mass range. Here we describe optimization of various instrument and post-acquisition parameters that collectively provide for quantification of iTRAQ-labeled phosphorylated peptides isolated from complex cell lysates. Peptides spanning a concentration dynamic range of 100:1 are readily quantified. Our results indicate that appropriate parameterization of collision energy as a function of precursor m/z and z provides for optimal performance in terms of peptide identification and relative quantification by iTRAQ. Using this approach, we readily identify activated signaling pathways downstream of oncogenic mutants of Flt-3 kinase in a model system of human myeloid leukemia. [4][5][6], extended mass range capability [7], use of helium buffer gas [3], and automated control of trapped ion populations [8], all combined to catapult trap-based instruments to the forefront of proteomics research. Concomitant advances in CPU and embedded systems provided for rapid and automated control of instrument parameters and yielded heretofore unattainable throughput for peptide sequence analysis via LC-MS/MS. Today, several years past the emergence of proteomics as a field of active research [9], it is clear that "typical" tryptic peptides are very amenable to sequence analysis via low-energy MS/MS. More recently, the introduction of hybrid geometries [10 -12] and other instrument configurations [13,14], along with protocols for enrichment of post-translationally modified peptides [15,16], have dramatically increased the experimental dynamic range of proteomics analyses. Collectively, these results have revealed that peptides decorated with labile modifications often are not well behaved under low-energy MS/MS conditions. For example, facile loss of phosphoric acid from the side chains of serine-and threoninephosphorylated peptides often limits the available sequence-specific information contained in associated MS/MS spectra [17,18]. Interestingly, recent reports [19,20] suggest that higher energy, or "triple quadrupole like," MS/MS can provide improved sequence analysis, particularly for phosphorylated peptides. In the work repo...