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Coumarin Tags for Analysis of Peptides by MALDI-TOF MS and MS/MS. 2. Alexa Fluor 350 Tag for Increased Peptide and Protein Identification by LC-MALDI-TOF/TOF MS

The goal of this study was the development of N-terminal tags to improve peptide identification using high-throughput MALDI-TOF/TOF MS. Part 1 of the study was focused on the influence of derivatization on the intensities of MALDI-TOF MS signals of peptides. In part 2, various derivatization approaches for the improvement of peptide fragmentation efficiency in MALDI-TOF/TOF MS are explored. We demonstrate that permanent cation tags, while significantly improving signal intensity in the MS mode, lead to severe suppression of MS/MS fragmentation, making these tags unsuitable for high-throughput MALDI-TOF/TOF MS analysis. In the present work, it was found that labeling with Alexa Fluor 350, a coumarin tag containing a sulfo group, along with guanidation of epsilon-amino groups of Lys, could enhance unimolecular fragmentation of peptides with the formation of a high-intensity y-ion series, while the peptide intensities in the MS mode were not severely affected. LC-MALDI-TOF/TOF MS analysis of tryptic peptides from the SCX fractions of an E. coli lysate revealed improved peptide scores, a doubling of the total number of peptides, and a 30% increase in the number of proteins identified, as a result of labeling. Furthermore, by combining the data from native and labeled samples, confidence in correct identification was increased, as many proteins were identified by different peptides in the native and labeled data sets. Additionally, derivatization was found not to impair chromatographic behavior of peptides. All these factors suggest that labeling with Alexa Fluor 350 is a promising approach to the high-throughput LC-MALDI-TOF/TOF MS analysis of proteomic samples.

Improved peptide identification in proteomics by two consecutive stages of mass spectrometric fragmentation.

MS-based proteomics usually involves the fragmentation of tryptic peptides (tandem MS or MS(2)) and their identification by searching protein sequence databases. In ion trap instruments fragments can be further fragmented and analyzed, a process termed MS/MS/MS or MS(3). Here, we report that efficient ion capture in a linear ion trap leads to MS(3) acquisition times and spectra quality similar to those for MS(2) experiments with conventional 3D ion traps. Fragmentation of N- or C-terminal ions resulted in informative and low-background spectra, even at subfemtomol levels of peptide. Typically C-terminal ions are chosen for further fragmentation, and the MS(3) spectrum greatly constrains the C-terminal amino acids of the peptide sequence. MS(3) spectra allow resolution of ambiguities in identification, a crucial problem in proteomics. Because of the sensitivity and rapid scan rates of the linear ion trap, several MS(3) spectra per peptide can be obtained even when sequencing very complex mixtures. We calculate the probability that an experimental MS(3) spectrum originates from fragmentation of a given N- or C-terminal ion of a peptide under consideration. This MS(3) identification score can be combined with the MS(2) scores of the precursor peptide from existing search engines. When MS(3) is performed on the linear ion trap-Fourier transform mass spectrometer combination, accurate peptide masses further increase confidence in peptide identification.

Coumarin tags for improved analysis of peptides by MALDI-TOF MS and MS/MS. 1. Enhancement in MALDI MS signal intensities.

The goal of this study was the development of N-terminal tags to improve peptide identification using high-throughput MALDI-TOF MS and MS/MS. The proposed tags, commercially available fluorescent derivatives of coumarin, can be advantageous for peptide analysis in both MS and MS/MS modes. This paper, part 1, will focus on the influence of derivatization on the intensities of MALDI-TOF MS signals of peptides. Labeling peptides with tags containing the coumarin core was found to enhance the intensities of peptide peaks (in some cases over 40-fold) in MALDI-TOF MS using CHCA and 2,5-DHAP matrixes. The signal enhancement was found to be peptide- and matrix-dependent, being the most pronounced for hydrophilic peptides. No correlation was found between the UV absorptivity of the tags at the excitation wavelengths typical for UV-MALDI and the magnitude of the signal enhancement. Interestingly, peptides labeled with Alexa Fluor 350, a coumarin derivative containing a sulfo group (i.e., bearing strong negative charge), showed a 5-15-fold increase in intensity of MALDI MS signal in the positive ion mode, relative to the underivatized peptides, when 2,5-DHAP was used as the matrix. The Alexa Fluor 350 tag yielded a significantly higher signal relative to that for the CAF tag, likely due to the increased hydrophobicity of the coumarin structure. With 2,5-DHB, a decrease of MALDI MS signal was observed for all coumarin-labeled peptides, again relative to the unlabeled species. These findings support the hypothesis that derivatization with coumarin, a relatively hydrophobic structure, improves incorporation of hydrophilic peptides into hydrophobic MALDI matrixes, such as CHCA and 2,5-DHAP.

Statistical characterization of ion trap tandem mass spectra from doubly charged tryptic peptides.

Collision-induced dissociation (CID) is a common ion activation technique used to energize mass-selected peptide ions during tandem mass spectrometry. Characteristic fragment ions form from the cleavage of amide bonds within a peptide undergoing CID, allowing the inference of its amino acid sequence. The statistical characterization of these fragment ions is essential for improving peptide identification algorithms and for understanding the complex reactions taking place during CID. An examination of 1465 ion trap spectra from doubly charged tryptic peptides reveals several trends important to understanding this fragmentation process. While less abundant than y ions, b ions are present in sufficient numbers to aid sequencing algorithms. Fragment ions exhibit a characteristic series-specific relationship between their masses and intensities. Each residue influences fragmentation at adjacent amide bonds, with Pro quantifiably enhancing cleavage at its N-terminal amide bond and His increasing the formation of b ions at its C-terminal amide bond. Fragment ions corresponding to a formal loss of ammonia appear preferentially in peptides containing Gln and Asn. These trends are partially responsible for the complexity of peptide tandem mass spectra.