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.
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 ϩ1-98'). 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. (J Am Soc Mass Spectrom 2010, 21, 2031-2040
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.
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.
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