Jason C. Rogalski

Mass spectrometric identification of formaldehyde-induced peptide modifications under in vivo protein cross-linking conditions

Formaldehyde cross-linking of proteins is emerging as a novel approach to study protein-protein interactions in living cells. It has been shown to be compatible with standard techniques used in functional proteomics such as affinity-based protein enrichment, enzymatic digestion, and mass spectrometric protein identification. So far, the lack of knowledge on formaldehyde-induced protein modifications and suitable mass spectrometric methods for their targeted detection has impeded the identification of the different types of cross-linked peptides in these samples. In particular, it has remained unclear whether in vitro studies that identified a multitude of amino acid residues reacting with formaldehyde over the course of several days are suitable substitutes for the much shorter reaction times of 10-20 min used in cross-linking experiments in living cells. The current study on model peptides identifies amino-termini as well as lysine, tryptophan, and cysteine side chains, i.e. a small subset of those modified after several days, as the major reactive sites under such conditions, and suggests relative position in the peptide sequence as well as sequence microenvironment to be important factors that govern reactivity. Using MALDI-MS, mass increases of 12 Da on amino groups and 30 Da on cysteines were detected as the major reaction products, while peptide fragment ion analysis by tandem mass spectrometry was used to localize the actual modification sites on a peptide. Non-specific cross-linking was absent, and could only be detected with low yield at elevated peptide concentrations. The detailed knowledge on the constraints and products of the formaldehyde reaction with peptides after short incubation times presented in this study is expected to facilitate the targeted mass spectrometric analysis of proteins after in vivo formaldehyde cross-linking.

Identification of Nitroxyl-induced Modifications in Human Platelet Proteins Using a Novel Mass Spectrometric Detection Method

Nitroxyl (HNO) exhibits many important pharmacological effects, including inhibition of platelet aggregation, and the HNO donor Angelis salt has been proposed as a potential therapeutic agent in the treatment of many diseases including heart failure and alcoholism. Despite this, little is known about the mechanism of action of HNO, and its effects are rarely linked to specific protein targets of HNO or to the actual chemical changes that proteins undergo when in contact with HNO. Here we study the presumed major molecular target of HNO within the body: protein thiols. Cysteine-containing tryptic peptides were reacted with HNO, generating the sulfinamide modification and, to a lesser extent, disulfide linkages with no other long lived intermediates or side products. The sulfinamide modification was subjected to a comprehensive tandem mass spectrometric analysis including MS/MS by CID and electron capture dissociation as well as an MS3 analysis. These studies revealed a characteristic neutral loss of HS(O)NH2 (65 Da) that is liberated from the modified cysteine upon CID and can be monitored by mass spectrometry. Upon storage, partial conversion of the sulfinamide to sulfinic acid was observed, leading to coinciding neutral losses of 65 and 66 Da (HS(O)OH). Validation of the method was conducted using a targeted study of nitroxylated glyceraldehyde-3-phosphate dehydrogenase extracted from Angelis salt-treated human platelets. In these ex vivo experiments, the sample preparation process resulted in complete conversion of sulfinamide to sulfinic acid, making this the sole subject of further ex vivo studies. A global proteomics analysis to discover platelet proteins that carry nitroxyl-induced modifications and a mass spectrometric HNO dose-response analysis of the modified proteins were conducted to gain insight into the specificity and selectivity of this modification. These methods identified 10 proteins that are modified dose dependently in response to HNO, whose functions range from metabolism and cytoskeletal rearrangement to signal transduction, providing for the first time a possible mechanistic link between HNO-induced modification and the physiological effects of HNO donors in platelets.

Mass spectrometry-based proteomics in biomedical research: emerging technologies and future strategies.

In recent years, the technology and methods widely available for mass spectrometry (MS)-based proteomics have increased in power and potential, allowing the study of protein-level processes occurring in biological systems. Although these methods remain an active area of research, established techniques are already helping answer biological questions. Here, this recent evolution of MS-based proteomics and its applications are reviewed, including standard methods for protein and peptide separation, biochemical fractionation, quantitation, targeted MS approaches such as selected reaction monitoring, data analysis and bioinformatics. Recent research in many of these areas reveals that proteomics has moved beyond simply cataloguing proteins in biological systems and is finally living up to its initial potential - as an essential tool to aid related disciplines, notably health research. From here, there is great potential for MS-based proteomics to move beyond basic research, into clinical research and diagnostics.

In Silico Protein Interaction Analysis Using the Global Proteome Machine Database

Experiments to probe for protein-protein interactions are the focus of functional proteomic studies, thus proteomic data repositories are increasingly likely to contain a large cross-section of such information. Here, we use the Global Proteome Machine database (GPMDB), which is the largest curated and publicly available proteomic data repository derived from tandem mass spectrometry, to develop an in silico protein interaction analysis tool. Using a human histone protein for method development, we positively identified an interaction partner from each histone protein family that forms the histone octameric complex. Moreover, this method, applied to the α subunits of the human proteasome, identified all of the subunits in the 20S core particle. Furthermore, we applied this approach to human integrin αIIb and integrin β3, a major receptor involved in the activation of platelets. We identified 28 proteins, including a protein network for integrin and platelet activation. In addition, proteins interacting with integrin β1 obtained using this method were validated by comparing them to those identified in a formaldehyde-supported coimmunoprecipitation experiment, protein-protein interaction databases and the literature. Our results demonstrate that in silico protein interaction analysis is a novel tool for identifying known/candidate protein-protein interactions and proteins with shared functions in a protein network.

Accessibility governs the relative reactivity of basic residues in formaldehyde-induced protein modifications

Cross-linking of proteins in a complex requires the chemical modification of the proteins in order to form a covalent link. This can be achieved in vivo using formaldehyde as it is small and rapidly permeates the cell membrane. Previous model studies of the speed and specificity of the first step of this reaction on peptides have suggested that residue accessibility and sequence micro-environment play a significant role in the production of the reactive intermediate necessary for cross-linking. This dependency was therefore further investigated on model proteins, which contain a more complex tertiary structure. Under mild reaction conditions, similar to those used for in vivo protein cross-linking, it was found that the vast majority of modification occurred on lysines, tertiary structure and solvent accessible surface area played a major role in regulating the extent of formaldehyde-induced modifications, and that the modifications on a folded protein did not significantly affect its tertiary structural stability.

Catabolism of the Last Two Steroid Rings in Mycobacterium tuberculosis and Other Bacteria

ABSTRACT Most mycolic acid-containing actinobacteria and some proteobacteria use steroids as growth substrates, but the catabolism of the last two steroid rings has yet to be elucidated. In Mycobacterium tuberculosis, this pathway includes virulence determinants and has been proposed to be encoded by the KstR2-regulated genes, which include a predicted coenzyme A (CoA) transferase gene (ipdAB) and an acyl-CoA reductase gene (ipdC). In the presence of cholesterol, ΔipdC and ΔipdAB mutants of either M. tuberculosis or Rhodococcus jostii strain RHA1 accumulated previously undescribed metabolites: 3aα-H-4α(carboxyl-CoA)-5-hydroxy-7aβ-methylhexahydro-1-indanone (5-OH HIC-CoA) and (R)-2-(2-carboxyethyl)-3-methyl-6-oxocyclohex-1-ene-1-carboxyl-CoA (COCHEA-CoA), respectively. A ΔfadE32 mutant of Mycobacterium smegmatis accumulated 4-methyl-5-oxo-octanedioic acid (MOODA). Incubation of synthetic 5-OH HIC-CoA with purified IpdF, IpdC, and enoyl-CoA hydratase 20 (EchA20), a crotonase superfamily member, yielded COCHEA-CoA and, upon further incubation with IpdAB and a CoA thiolase, yielded MOODA-CoA. Based on these studies, we propose a pathway for the final steps of steroid catabolism in which the 5-member ring is hydrolyzed by EchA20, followed by hydrolysis of the 6-member ring by IpdAB. Metabolites accumulated by ΔipdF and ΔechA20 mutants support the model. The conservation of these genes in known steroid-degrading bacteria suggests that the pathway is shared. This pathway further predicts that cholesterol catabolism yields four propionyl-CoAs, four acetyl-CoAs, one pyruvate, and one succinyl-CoA. Finally, a ΔipdAB M. tuberculosis mutant did not survive in macrophages and displayed severely depleted CoASH levels that correlated with a cholesterol-dependent toxicity. Our results together with the developed tools provide a basis for further elucidating bacterial steroid catabolism and virulence determinants in M. tuberculosis. IMPORTANCE Bacteria are the only known steroid degraders, but the pathway responsible for degrading the last two steroid rings has yet to be elucidated. In Mycobacterium tuberculosis, this pathway includes virulence determinants. Using a series of mutants in M. tuberculosis and related bacteria, we identified a number of novel CoA thioesters as pathway intermediates. Analysis of the metabolites combined with enzymological studies establishes how the last two steroid rings are hydrolytically opened by enzymes encoded by the KstR2 regulon. Our results provide experimental evidence for novel ring-degrading enzymes, significantly advance our understanding of bacterial steroid catabolism, and identify a previously uncharacterized cholesterol-dependent toxicity that may facilitate the development of novel tuberculosis therapeutics. Bacteria are the only known steroid degraders, but the pathway responsible for degrading the last two steroid rings has yet to be elucidated. In Mycobacterium tuberculosis, this pathway includes virulence determinants. Using a series of mutants in M. tuberculosis and related bacteria, we identified a number of novel CoA thioesters as pathway intermediates. Analysis of the metabolites combined with enzymological studies establishes how the last two steroid rings are hydrolytically opened by enzymes encoded by the KstR2 regulon. Our results provide experimental evidence for novel ring-degrading enzymes, significantly advance our understanding of bacterial steroid catabolism, and identify a previously uncharacterized cholesterol-dependent toxicity that may facilitate the development of novel tuberculosis therapeutics.

A New Ion Source and Procedures for Atmospheric Pressure-Electron Capture Dissociation of Peptides

We introduce a new atmospheric pressure-electron capture dissociation (AP-ECD) source in which conventional nanospray emitters are coupled with the source block and photoionization lamp of a PhotoSpray APPI source. We also introduce procedures for data collection and processing, aimed at maximizing the signal-to-background ratio of ECD products. Representative data from Substance P are presented to demonstrate the performance of the technique. Further, we demonstrate the effects of two important experimental variables, source temperature and vacuum-interface declustering potential (DP), on the method. Last, we show that even when a high source temperature is used to maximize efficiency, AP-ECD fragments of a model phosphorylated peptide retain the modification.

Atmospheric pressure-electron capture dissociation of peptides using a modified PhotoSpray ion source.

An improved in-source atmospheric pressure-electron capture dissociation (AP-ECD) method is described. Building upon the early example of Laprévotes group, photoelectrons generated within a commercial PhotoSpray atmospheric pressure photoionization source are used to induce ECD of multiply charged peptide ions originating from an upstream heated nebulizer device. To attain high sensitivity, the method makes use of a novel electropneumatic-heated nebulizer to assist in the creation and transmission of multiply charged ions from sample solutions. Here, we demonstrate that readily interpretable AP-ECD spectra of infused peptides can be acquired from 100 fmol sample consumed, on a chromatographic time scale, using a conventional quadrupole time-of-flight (Q-ToF) mass spectrometer otherwise incapable of ECD/ETD experiments. Though much work remains to be done to develop and characterize the method, the results indicate that AP-ECD has the potential to be a practical new tool for the mass spectrometric analysis of peptides and proteins.

Identification and characterization of seed coat mucilage proteins

Arabidopsis seed coat mucilage, an extracellular matrix composed of cell wall carbohydrates, contains a proteome functionally similar to that of cell wall but also includes proteins unique to mucilage. Plant cell wall proteins are important regulators of cell wall architecture and function. However, because cell wall proteins are difficult to extract and analyze, they are generally poorly understood. Here, we describe the identification and characterization of proteins integral to the Arabidopsis (Arabidopsis thaliana) seed coat mucilage, a specialized layer of the extracellular matrix composed of plant cell wall carbohydrates that is used as a model for cell wall research. The proteins identified in mucilage include those previously identified by genetic analysis, and several mucilage proteins are reduced in mucilage-deficient mutant seeds, suggesting that these proteins are genuinely associated with the mucilage. Arabidopsis mucilage has both nonadherent and adherent layers. Both layers have similar protein profiles except for proteins involved in lipid metabolism, which are present exclusively in the adherent mucilage. The most abundant mucilage proteins include a family of proteins named TESTA ABUNDANT1 (TBA1) to TBA3; a less abundant fourth homolog was named TBA-LIKE (TBAL). TBA and TBAL transcripts and promoter activities were detected in developing seed coats, and their expression requires seed coat differentiation regulators. TBA proteins are secreted to the mucilage pocket during differentiation. Although reverse genetics failed to identify a function for TBAs/TBAL, the TBA promoters are highly expressed and cell type specific and so should be very useful tools for targeting proteins to the seed coat epidermis. Altogether, these results highlight the mucilage proteome as a model for cell walls in general, as it shares similarities with other cell wall proteomes while also containing mucilage-specific features.

Liquid chromatography-atmospheric pressure electron capture dissociation mass spectrometry for the structural analysis of peptides and proteins.

Atmospheric pressure electron capture dissociation (AP-ECD) is an emerging technique with the potential to be a more accessible alternative to conventional ECD/electron transfer dissociation (ETD) methods because it can be implemented using a stand-alone ion source device suitable for use with any existing or future electrospray ionization mass spectrometer. With AP-ECD, no modification of the main instrument is required, so it may easily be retrofitted to instruments not originally equipped with ECD/ETD capabilities. Here, we present our first purpose-built AP-ECD source and demonstrate its use in conjunction with capillary LC for the analysis of substance P, a tryptic digest of bovine serum albumin, and a phosphopeptide mixture. Quality ECD spectra were obtained for all the samples at the low femtomole level, proving that LC-AP-ECD-MS is suitable for the structural analysis of peptides and protein digests, in this case using an unmodified quadrupole time-of-flight mass spectrometer built ca. 2002.