Femtosecond laser-induced ionization/dissociation tandem mass spectrometry (fsLID-MS/MS) of deprotonated phosphopeptide anions.

RATIONALE: Radical-directed dissociation techniques provide structural information which is complementary to that from conventional collision-induced dissociation (CID). The analysis of phosphopeptide anions is warranted due to their relatively acidic character. As femtosecond laser-induced ionization/dissociation tandem mass spectrometry (fsLID-MS/MS) is uniquely initiated by field ionization, an investigation is warranted to determine whether fsLID may provide novel analytical utility for phosphopeptide anions. METHODS: Twenty-three synthetic deprotonated phosphopeptide anions were introduced into a three-dimensional quadrupole ion trap mass spectrometer via electrospray ionization. The ion trap was interfaced with a near-IR (802 nm) ultrashort-pulsed (35 fs FWHM) ultrahigh-powered (peak power ~10(14) W/cm(2)) laser system. Performance comparisons are made with other techniques applied to phosphopeptide anion analysis, including CID, electron detachment dissociation (EDD), negative electron transfer dissociation (NETD), activated electron photodetachment dissociation (activated-EPD), and ultraviolet photodissociation (UVPD). RESULTS: FsLID-MS/MS of multiply deprotonated phosphopeptide anions provides sequence information via phosphorylation-intact a/x ions in addition to other sequence ions, satellite ions, and side-chain losses. Novel fragmentation processes include selective c-ion formation N-terminal to Ser/Thr and a phosphorylation-specific correlation between xn -98 ion abundances and phosphorylation at the n(th) residue. Sequencing-quality data required about 30 s of signal averaging. fsLID-MS/MS of singly deprotonated phosphopeptides did not yield product anions with stable trajectories, despite significant depletion of the precursor. CONCLUSIONS: Multiply deprotonated phosphopeptide anions were sequenced via negative-mode fsLID-MS/MS, with phosphosite localization facilitated by a/x ion series in addition to diagnostic x(n)-98 ions. fsLID-MS/MS is qualitatively competitive with other techniques. Further efficiency enhancements (e.g., implementation on a linear trap or/and higher pulse frequencies) may permit sequence analyses on chromatographic timescales.

Mechanism elucidation for nonstochastic femtosecond laser-induced ionization/dissociation: from amino acids to peptides.

Femtosecond laser-induced ionization/dissociation (fs-LID) has been demonstrated as a novel ion activation method for use in tandem mass spectrometry. The technique opens the door to unique structural information about biomolecular samples that is not easily accessed by traditional means. fs-LID is able to cleave strong bonds while keeping weaker bonds intact. This feature has been found to be particularly useful for the mapping of post-translational modifications such as phosphorylation, which is difficult to achieve by conventional proteomic studies. Here we investigate the laser-ion interaction on a fundamental level through the characterization of fs-LID spectra for the protonated amino acids and two series of derivatized samples. The findings are used to better understand the fs-LID spectra of synthetic peptides. This is accomplished by exploring the effects of several single-residue substitutions.

Tandem mass spectrometry strategies for phosphoproteome analysis.

Protein phosphorylation is involved in nearly all essential biochemical pathways and the deregulation of phosphorylation events has been associated with the onset of numerous diseases. A multitude of tandem mass spectrometry (MS/MS) and multistage MS/MS (i.e., MS(n) ) strategies have been developed in recent years and have been applied toward comprehensive phosphoproteomic analysis, based on the interrogation of proteolytically derived phosphopeptides. However, the utility of each of these MS/MS and MS(n) approaches for phosphopeptide identification and characterization, including phosphorylation site localization, is critically dependant on the properties of the precursor ion (e.g., polarity and charge state), the specific ion activation method that is employed, and the underlying gas-phase ion chemistries, mechanisms and other factors that influence the gas-phase fragmentation behavior of phosphopeptide ions. This review therefore provides an overview of recent studies aimed at developing an improved understanding of these issues, and highlights the advantages and limitations of both established (e.g., CID) and newly maturing (e.g., ECD, ETD, photodissociation, etc.) yet complementary, ion activation techniques. This understanding is expected to facilitate the continued refinement of existing MS/MS strategies, and the development of novel MS/MS techniques for phosphopeptide analysis, with great promise in providing new insights into the role of protein phosphorylation on normal biological function, and in the onset and progression of disease. © 2011 Wiley Periodicals, Inc., Mass Spec Rev 30:600-625, 2011.

Enhanced characterization of singly protonated phosphopeptide ions by femtosecond laser-induced ionization/dissociation tandem mass spectrometry (fs-LID-MS/MS).

To develop an improved understanding of the regulatory role that post-translational modifications (PTMs) involving phosphorylation play in the maintenance of normal cellular function, tandem mass spectrometry (MS/MS) strategies coupled with ion activation techniques such as collision-induced dissociation (CID) and electron-transfer dissociation (ETD) are typically employed to identify the presence and site-specific locations of the phosphate moieties within a given phosphoprotein of interest. However, the ability of these techniques to obtain sufficient structural information for unambiguous phosphopeptide identification and characterization is highly dependent on the ion activation method employed and the properties of the precursor ion that is subjected to dissociation. Herein, we describe the application of a recently developed alternative ion activation technique for phosphopeptide analysis, termed femtosecond laser-induced ionization/dissociation (fs-LID). In contrast to CID and ETD, fs-LID is shown to be particularly suited to the analysis of singly protonated phosphopeptide ions, yielding a wide range of product ions including a, b, c, x, y, and z sequence ions, as well as ions that are potentially diagnostic of the positions of phosphorylation (e.g., 'a(n)+1-98'). Importantly, the lack of phosphate moiety losses or phosphate group 'scrambling' provides unambiguous information for sequence identification and phosphorylation site characterization. Therefore, fs-LID-MS/MS can serve as a complementary technique to established methodologies for phosphoproteomic analysis.

Applications of femtochemistry to proteomic and metabolomic analysis.

Femtosecond laser pulses have been widely used as a tool to study molecular ionization and fragmentation. This article bridges the application of femtosecond laser technology in early research focused on small isolated molecules with that in modern biological mass spectrometry for proteomics and metabolomic analysis on large (140+ atoms) biomolecules. The single-shot interaction of a femtosecond laser with neutral para-nitrotoluene (pNT) is investigated with time-of-flight mass spectrometry and compared with the ultrafast photodissociation of protonated pNT in an ion trap mass spectrometer accumulated over ∼1000 pulses. The ion trap experiment is then extended to longer biomolecules. As demonstrated in the examples of vasopressin and tomatine, this novel ion activation method provides greater sequence coverage and nonstatistical fragmentation, leading to valuable information complementary to conventional methods for structural analysis.

Femtosecond laser-induced ionization/dissociation of protonated peptides.

Although tandem mass spectrometry has revolutionized the identification and structural characterization of peptides and proteins, future advances in comprehensive proteome analysis will depend on the development of improved methods for ion activation that yield greater sequence information, and with selective control over the fragmentation chemistry. This report presents initial findings that demonstrate the utility of a novel ion activation method using ultrashort (approximately 30 fs) laser pulses as a means to overcome the limitations of current technologies, while opening the door to solving significant challenges in protein and peptide analysis.