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Chemical cross-linking coupled with mass spectrometry plays an important role in unravelling protein interactions, especially weak and transient ones. Moreover, cross-linking complements several structural determination approaches such as cryo-EM. Although several computational approaches are available for the annotation of spectra obtained from cross-linked peptides, there remains room for improvement. Here, we present Xilmass, a novel algorithm to identify cross-linked peptides that introduces two new concepts: (i) the cross-linked peptides are represented in the search database such that the cross-linking sites are explicitly encoded, and (ii) the scoring function derived from the Andromeda algorithm was adapted to score against a theoretical tandem mass spectrometry (MS/MS) spectrum that contains the peaks from all possible fragment ions of a cross-linked peptide pair. The performance of Xilmass was evaluated against the recently published Kojak and the popular pLink algorithms on a calmodulin-plectin complex data set, as well as three additional, published data sets. The results show that Xilmass typically had the highest number of identified distinct cross-linked sites and also the highest number of predicted cross-linked sites.

An approach is tested here as a rapid screening method for Bacillus spore species employing bacterial peptide analysis with a miniaturized MALDI TOF mass spectrometer. A limited set of tryptic peptides was generated in situ following selective solubilization of the small, acid-soluble protein family (SASP) from spore samples on the MALDI sample holder. To facilitate species identification, a compact database was created comprising masses of the tryptic cleavage products generated in silico from all Bacillus and Clostridium SASPs whose sequences are available in public databases. Experimental measurements were matched against the custom-made database, and a published statistical model was then used to evaluate the probability of false identifications.

A novel ion trap time-of-flight hybrid mass spectrometer (qIT-TOF MS) has been applied for peptide sequencing in proteolytic digests generated from spore mixtures of Bacilli. The method of on-probe solubilization and in situ proteolytic digestion of small, acid-soluble spore proteins has been recently developed in our laboratory, and microorganism identification in less than 20 min was accomplished. In this study, tryptic peptides were generated in situ from complex spore mixtures of B. subtilis 168, B. globigii, B. thuringiensis subs. Kurstaki, and B. cereus T, respectively. MALDI analysis of bacterial peptides generated was performed with an average mass resolving power of 6200 and a mass accuracy of up to 10 ppm using a trap-TOF tandem configuration. Precursor ions of interest were usually selected and stored in the quadrupole ion trap with their complete isotope distribution by choosing a window of +/- 2 Da. Sequence-specific information on isolated protonated peptides was gained via tandem MS experiments with an average mass resolving power of 4450 for product ion analysis, and protein and bacterial sources were identified by database searching.

A method to investigate the chemical composition of organic aerosols formed from biogenic hydrocarbon oxidation using atmospheric pressure chemical ionization mass spectrometry (APCI/MS) is described. The method involves the direct introduction of aerosol particles into the ion source of the mass spectrometer. Using this technique, reaction monitoring experiments of alpha-pinene ozonolysis show the formation of hetero- and homomolecular cluster anions (dimers) of the primary oxidation products (multifunctional carboxylic acids). Since the formation of dimers plays a profound role in new particle formation processes by homogeneous nucleation in the atmosphere and, at the same time, is an intrinsic feature of APCI, it is essential to differentiate between both processes when on-line APCI/MS is applied. In this paper, we compare the results from the investigations of organic aerosols and artificially generated dimer cluster ions of the same compounds using identical ionization conditions. The clusters and their formation processes are characterized by varying the analyte concentration, investigating the thermal stability of dimers, and studying collisional activation properties of both ion species. The investigations show a significant difference in ion stability: dimer anions measured on-line have an estimated stability that is 20 kJ mol(-1) higher than that of the corresponding artificially generated cluster ions. Hence, the technique provides the possibility to accurately characterize dimers as ionized reaction products from biogenic hydrocarbon oxidation and allows an insight into the process of new-particle formation by homogeneous nucleation.

A strategy is proposed for the rapid identification of Bacillus spores, which relies on the selective release of a family of proteins, referred to as small, acid-soluble spore proteins (SASPs). In this work, SASPs were selectively solubilized from Bacillus spores on the MALDI sample plate by using 10% TFA. Proteolytic digests of SASPs generated in situ from spores of B. subtilis 168, B. globigii, B. thuringiensis subs. Kurstaki HD-1, B. cereus T, and the nonpathogenic strain B. anthracis Sterne were prepared in 5-25 min by using trypsin immobilized on Agarose beads and subsequently analyzed by MALDI-TOFMS using a curved-field reflectron. Protein identification was obtained by partial sequencing of distinctive tryptic peptides from Bacillus spores via post-source decay analysis combined with genome-based database searches by Mascot Sequence Query. Various unique SASPs were identified, allowing the characterization of Bacillus species by obtaining sequence-specific information on single peptides. The applicability of this approach for the rapid identification of Bacillus species was further established by analyzing spore mixtures.

An approach is tested here as a rapid screening method for Bacillus spore species employing bacterial peptide analysis with a miniaturized MALDI TOF mass spectrometer. A limited set of tryptic peptides was generated in situ following selective solubilization of the small, acid-soluble protein family (SASP) from spore samples on the MALDI sample holder. To facilitate species identification, a compact database was created comprising masses of the tryptic cleavage products generated in silico from all Bacillus and Clostridium SASPs whose sequences are available in public databases. Experimental measurements were matched against the custom-made database, and a published statistical model was then used to evaluate the probability of false identifications.

A slightly modified atmospheric pressure chemical ionization source is employed for direct quantitative analysis of volatile or semivolatile organic compounds in air. The method described here is based on the direct introduction of an analyte in the gas or particle phase, or both, into the ion source of a commercial ion trap mass spectrometer. For quantitation, a standard solution is directly transferred into the vaporizer unit of the ion source via a deactivated fused-silica capillary by using the sheath liquid syringe pump, which is part of the mass spectrometer. The standard addition procedure is conducted by varying the pump rate of a diluted solution of the standard compound in methanol/water. A N2 sheath gas flow is applied for optimal vaporization and mixing with the analyte gas stream. By performing detailed reagent ion monitoring experiments, it is shown that the relative signal intensity of [M + H]+ ions is dependent on the relative humidity of the analyte gas stream as well as the composition and concentration of CI reagent ions. The method is validated by a comparison of the standard addition results with a calibration test gas of known concentration. To demonstrate the potential of atmospheric pressure chemical ionization mass spectrometry as a quantitative analytical technique for on-line investigations, a tropospherically relevant reaction is carried out in a 493-L reaction chamber at atmospheric pressure and 296 K in synthetic air at 50% relative humidity. Finally, the applicability of the technique to rapidly differentiate between analytes in the gas and particle phase is demonstrated.

Knowledge about the functions of individual proteins on a system-wide level is crucial to fully understand molecular mechanisms underlying cellular processes. A considerable part of the proteome across all organisms is still poorly characterized. Mass spectrometry is an efficient technology for the global study of proteins. One of the most prominent methods for accurate proteome-wide comparative quantification is stable isotope labeling by amino acids in cell culture (SILAC). However, application of SILAC to prototrophic organisms such as Saccharomyces cerevisiae, also known as baker’s yeast, is compromised since they are able to synthesize all amino acids on their own. Here, we describe an advanced strategy, termed 2nSILAC, that allows for in vivo labeling of prototrophic baker’s yeast using heavy arginine and lysine under fermentable and respiratory growth conditions, making it a suitable tool for the global study of protein functions. This generic 2nSILAC strategy allows for directly using and systematically screening yeast mutant strain collections available to the scientific community. We exemplarily demonstrate its high potential by analyzing the effects of mitochondrial gene deletions in mitochondrial fractions using quantitative mass spectrometry revealing the role of Coi1 for the assembly of cytochrome c oxidase (respiratory chain complex IV).