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Rationale: Selective derivatization of peptide N-terminus with 4-formyl-benzenesulfonic acid (FBSA) enables chemically activated fragmentation in positive and negative ion modes (ESI+/−) under charge reduction conditions. Overlapped positive and negative tandem mass spectra show b-ions making the assignment of b-ion series fragments easy and accurate.// Methods: We developed an FBSA–peptide microwave-assisted derivatization procedure. Derivatized and nonderivatized bovine serum albumin tryptic peptides and insulin non-tryptic peptide were compared after tandem mass spectrometry (MS/MS) analysis in positive and negative ion modes. A high-quality data set of sulfonated b-ions obtained in negative tandem mass spectra of singly charged FBSA–peptides were matched to detected b-ions in positive MS/MS spectra. Moreover, negative spectra signals were converted and matched against y-ions in positive tandem mass spectra to identify complete peptide sequences.// Results: The FBSA derivatization procedure produced a significantly improved MS/MS data set (populated by high-intensity signals of b- and y-ions) compared to commonly used N-terminal sulfonation reagents. Undesired side reactions almost do not occur, and the procedure reduces the derivatization time. It was found that b-ion intensities comprise 15% and 13% compared to combined ion intensities generated in positive- and negative ion modes, respectively. High visibility of b- ion series in negative ion mode can be attributed to N-terminal sulfonation that had no negative effect on the production of b- and y-ion series in positive ion mode.// Conclusions: The FBSA derivatization and de novo sequencing approach outlined here is a reliable method for accurate peptide sequence assignment. Increased production of b-ions in positive- and negative ion modes greatly improves peak assignment and thus enables accurate sequence reconstruction. Implementation of the named methodology would improve the quality of de novo sequencing data and reduce the number of misinterpreted spectra.

RATIONALE One of the most challenging tasks of proteomics is peptide de novo sequencing. 4- Sulfophenyl isothiocyanate (SPITC) peptide derivatization enables acquisition of high- quality tandem mass spectra (MS/MS) for de novo sequencing, but unwanted non-specific reactions and reduced mass spectra (MS) signal intensities still represent the obstacles in high-throughput de novo sequencing. METHODS We developed a SPITC peptide derivatization procedure under acidic conditions (pH ≤5). Derivatized peptides were analyzed by matrix- assisted laser desorption/ionization (MALDI-MS) in negative ion mode followed by MS/MS in positive ion mode. A de novo sequencing tool, named DUST, adjusted to SPITC chemistry, was designed for successful high-throughput peptide de novo sequencing. This high-throughput peptide de novo sequencing was tested on Fusarium delphinoides, an organism with an uncharacterized genome. RESULTS The SPITC derivatization procedure under acidic conditions produced a significantly improved MS dataset in comparison to commonly used derivatization under basic conditions. Signal intensities were 6 to 10 times greater and the over-sulfonation effect measured on lysine- containing peptides was significantly decreased. Furthermore, development of a novel DUST algorithm enabled automated de novo sequencing with the calculated accuracy of 70.6%. CONCLUSIONS The SPITC derivatization and de novo sequencing approach outlined here provides a reliable method for high-throughput peptide de novo sequencing. High-throughput peptide de novo sequencing enabled protein mutation identification and identification of proteins from organisms with non-sequenced genomes.

RATIONALE We describe a novel negative chemically activated fragmentation/positive chemically activated fragmentation (CAF-/CAF+) technique for protein identification. The technique was used to investigate Lactobacillus brevis adaptation to nutrient deprivation. METHODS The CAF-/CAF+ method enables de novo sequencing of derivate peptides with negative and positive ion mode matrix-assisted laser desorption/ionization (MALDI) tandem mass spectrometry (MS/MS). Peptide sequences obtained from MS/MS spectra were matched against the National Center for Biotechnology Information (NCBI) non-redundant (nr) database and confirmed by the mass spectrometry data of elucidated peptide mass sequences derived from the annotated genome. This improved protein identification method highlighted 36 differentially expressed proteins in the proteome of L. brevis after 75 days of starvation. RESULTS The results revealed the key differences in the metabolic pathways that are responsible for the survival of L. brevis in a hostile environment. Proteomics analysis demonstrated that numerous proteins engaged in glucose and amino-acid catabolizing pathways, glycerolipid metabolizing pathways, and stress-response mechanisms are differentially expressed after long-term starvation. Amino acid and proteomics analysis indicated that starved L. brevis metabolized arginine, glycine, and histidine from dead cells as alternative nutrient sources. The production of lactic acid also varied between the parent cells and the starved cells. CONCLUSIONS Differentially expressed proteins identified exclusively by peptide sequence reading provided promising results for CAF-/CAF+ implementation in a standard proteomics workflow (e.g., biomarker and mutation discovery and biotyping). The practical performance of a reliable de novo sequencing technique in routine proteomics analysis is emphasized in this article. Copyright © 2013 John Wiley & Sons, Ltd. Copyright

Background The αv-integrin binding motif RGD4C (CDCRGDCFC) has been used extensively to circumvent inefficient adenovirus type 5 (Ad5) transduction of cells expressing low levels of the coxsackie and adenovirus receptor. However, until now, it has been unclear whether disulfide bonds in the RGD4C motif influence the retargeting potential of RGD4C-modified Ad5. Methods Replication deficient Ad5 bearing wild-type fiber (Ad5wt) or RGD4G, RGD4C and RGD2C2G insertions within the HI loop of the fiber protein (Ad5RGD4G, Ad5RGD4C and Ad5RGD2C2G, respectively) were used to transduce a panel of cancer cell lines, with or without previous treatment of these Ad5s with the reducing agent dithiothreitol (DTT). In parallel, native and DTT-treated fiber proteins isolated from purified Ad5RGD4C were compared by mass spectrometry. Results Ad5RGD4C transduced all studied cell lines much more efficiently than Ad5wt, whereas Ad5RGD4G transduced cells only slightly more efficiently than Ad5wt. DTT treatment had no effect on cell transduction by wild-type Ad5wt and Ad5RGD4G but abolished the increased transduction efficacy of Ad5RGD4C in a dose-dependent manner. The mass spectra of native and DTT-reduced tryptic digests of the Ad5RGD4C fiber protein are consistent with the presence of a C547–C549 linkage in the C547DC549RGDC553FC555 motif. Finally, the high transduction efficacy of Ad5RGD4C is conserved in Ad5RGD2C2G. Conclusions We provide genetic and biochemical data strongly suggesting that cysteines C547 and C549 from the C547DC549RGDC553FC555 motif inserted in the HI loop of the Ad5 fiber form a single disulfide bond, with this disulfide bond being crucial for Ad5RGD4C retargeting to av-integrins. Copyright © 2012 John Wiley & Sons, Ltd.

S-Adenosylhomocysteine hydrolase (AHCY) deficiency is a rare congenital disorder in methionine metabolism clinically characterized by white matter atrophy, delayed myelination, slowly progressive myopathy, retarded psychomotor development and mildly active chronic hepatitis. In the present study, we utilized a comparative proteomics strategy based on 2-DE/MALDI-MS and LC/ESI-MS to analyze plasma proteins from three AHCY-deficient patients prior to and after receiving dietary treatment designed to alleviate disease symptoms. Obtained results revealed candidate biomarkers for the detection of myopathy specifically associated with AHCY deficiency, such as carbonic anhydrase 3, creatine kinase, and thrombospondin 4. Several proteins mediating T-cell activation and function were identified as well, including attractin and diacylglycerol kinase α. Further validation and functional analysis of identified proteins with clinical value would ensure that these biomarkers make their way into routine diagnosis and management of AHCY deficiency.

The combination of separation techniques and mass spectrometry (MS) for peptide investigation allows superior sensitivity of detection and richer fragmentation data than available by direct MS analysis of a complex mixture. In this regard, liquid chromatography (LC) coupled to electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) MS have evolved as versatile analytical tools in proteomics. Very often, however, the product ion mass spectrum is either incomplete or overfilled with ions, thus making sequence analysis difficult. Here we report overall ion intensity improvement of C-terminal lysine-containing peptides from Lys-C digest by on-column derivatization of lysines with 2-methoxy-4, 5-dihydro-1H-imidazole. The method is simple, fast and exhibits 100% efficiency of the reaction. Additionally, post-source decay carried out on derivatized peptides gave rise almost exclusively to y-series ion formation, at 100% sequence coverage and high intensity. The novelty of the method resides in the side reaction of this derivatization process, namely the methylation of cysteines. This facilitates the estimation of the disulfide bridge position in a protein and the fragmentation of cysteine-containing peptide fragments. Additionally, by using this derivatization procedure, the loss of peptides, their degradation and/or oxidation, usually occurring in digest alkylation procedures, is greatly minimized. The new on-column derivatization protocol is designed to be carried out on C-18 Spin Tubes or Cleanup C18 Pipette Tips. We observed that use of buffered D2O solvent prevented unwanted oxidation and degradation reactions with respect to the stationary phase. This may be due to the fact that a deuteron is less polar than a proton, and thus the bonded silica stationary phase saturated with deuterons does not affect the reaction between epsilon-amino or cysteine thiol groups and 2-methoxy-4, 5-dihydro-1H- imidazole. Complete tagging of the peptides by on-column reaction could be obtained when using D2O, as compared to water-based reaction. Methylation of cysteine residues was enhanced when beta-mercaptoethanol was added in the reactant solution.

The usefulness of applying an integrated LC-NMR and LC-MS approach to acarbose bulk drug impurity profiling is demonstrated. LC-MS and LC-NMR methodologies were employed for the on-line separation and structural elucidation of a final drug product. Combining data provided by the stop-flow LC-NMR and LC-MS experiments made it possible to identify the main components present in the acarbose sample. Spectral analysis revealed that A and B were known impurities while C was an unknown compound. LC-MS and LC-NMR analyses revealed that C was a pentasacharide differing from the acarbose in a number and nature of sugar subunits in the molecule. It was subsequently isolated and its structure was confirmed by the off-line one- and two-dimensional NMR experiments and atom assignment was made.