Post-source Decay Research Articles

Mass spectrometric identification of multicage fullerene cycloadducts

Multicage fullerene cycloadducts have been detected by MALDI mass spectrometry; they have been found as admixtures in the products of reactions of the trifluoromethylation of fullerene samples doped with metallic sodium, the reaction between fullerenes and a mixture of trifluoromethylfullerenes, and the synthesis of fulleroproline esters. As a result, over 75 new compounds of this type have been identified. The optimization of the synthesis procedures and chromatographic fractionation allowed us to extract five compounds in the pure form: (C60)2(CF2)2(CF3)8, (C60)2(CF2)2(CF3)3C2F5, (C60)2(CF2)2(CF3)5C2F5, (C60)2(CF2)2(CF3)2O, and C60CH2N(CH2C60)CCOOtBu. Chemical structures of two of them, proposed on the basis of post source decay mass spectra, have been further confirmed by NMR spectra.

Mass Spectrometric Characterization of Modifications to Angiotensin II by Lipid Peroxidation Products, 4-Oxo-2(E)-nonenal and 4-Hydroxy-2(E)-nonenal

The octapeptide angiotensin II (Ang II; Asp(1)-Arg(2)-Val(3)-Tyr(4)-Ile(5)-His(6)-Pro(7)-Phe(8)) is the primary active hormone of the renin/angiotensin system (RAS) and has been implicated in various cardiovascular diseases. Numerous structure-activity relationship studies have identified Asp(1), Arg(2), and His(6) of Ang II to be critical for its biological activity and receptor binding. From the reactions of Ang II with lipid peroxidation-derived aldehydes, 4-oxo-2(E)-nonenal (ONE) or 4-hydroxy-2(E)-nonenal (HNE), we have identified the major modifications to the N-terminus, Asp(1), Arg(2), and His(6) of Ang II by liquid chromatography/mass spectrometry (LC/MS) and matrix-assisted laser desorption ionization-time-of-flight/MS (MALDI-TOF/MS). The identities of ONE- and HNE-modified Ang II were confirmed by tandem mass spectrometry (MS/MS) and postsource decay (PSD)-TOF/MS before and after the reaction with sodium borohydride. In the reaction with ONE, a pyruvamide-Ang II that formed via oxidative decarboxylation of N-terminal Asp was detected as the most abundant product after 48 h of incubation. It was followed by Arg-modified [Arg(2)(ONE-H(2)O)]-Ang II and the N-terminal-modified 4-ketoamide form of [N-ONE]-Ang II. The Michael addition products of [His(6)(HNE)]-Ang II were the most abundant products in the beginning of the reaction with HNE, followed by the dehydrated Michael addition products of [His(6)(HNE-H(2)O)]-Ang II. [His(6)(HNE)]-Ang II was dehydrated to [His(6)(HNE-H(2)O)]-Ang II during the prolonged incubation, and [His(6)(HNE-H(2)O)]-Ang II became the major products after 7 days. The model reactions of N(α)-tert-butoxycarbonyl (tBoc)-Arg with ONE and tBoc-His with HNE were performed and compared with the Ang II reaction. tBoc-Arg readily reacted with ONE to produce a compound analogous to [Arg(2)(ONE-H(2)O)]-Ang II, which confirmed Arg as one of the important target nucleophiles of ONE. However, tBoc-His exclusively formed a Michael addition product upon the reaction with HNE. The unexpected formation of [His(6)(HNE-H(2)O)]-Ang II can be explained by the proximity of His(6) to C-terminal carboxylate in the specific conformation of Ang II, which facilitates the dehydration of Michael addition products. Therefore, our results suggest a possible discrepancy in the adduction chemistry of ONE and HNE for model amino acids and endogenous bioactive peptides, which is governed by the microenvironment of peptides, such as the specific amino acid sequence and conformation. Such stable ONE- and HNE-derived modifications to Ang II could potentially modulate its functions in vivo by disrupting the interaction with Ang II type 1 (AT(1)) receptor and/or inhibiting the enzyme activity of aminopeptidase A (APA), which cleaves the N-terminal Asp residue of Ang II to generate Ang III.

Differentiation and Semiquantitative Analysis of an Isoaspartic Acid in Human α-Crystallin by Postsource Decay in a Curved Field Reflectron

Alpha-Crystallin, which forms a huge multimeric complex that is essential for maintaining eye lens transparency, is one of the major proteins in the lens. The protein, which exists as isoforms alphaA and alphaB, functions as a molecular chaperone to restore the original conformations of distorted constituent proteins in the lens. This function is important to maintain the transparency of the lens, because there is no protein turnover in the lens. Abnormal aggregation of constituent proteins in the lens has been reported in cataract patients, and deamidation of Asn as well as racemization and isomerization of Asp have been found in the alpha-Crystallin of these patients. While the establishment of a quick and facile analytical method is eagerly anticipated to investigate the relevance of the isomerization to pathological states such as cataracts, differentiating the isomerization states is still not performed routinely. Here, we report the differentiation and semiquantitative analysis of an isoaspartic acid (betaAsp) in human alpha-Crystallin using postsource decay on a MALDI-TOF mass spectrometer incorporating a curved field reflectron. Our reproducible results of analyzing synthetic and tryptic peptides containing betaAsp corroborated results obtained using a previously reported diagnostic ion, y(l-n+1) - 46, for the differentiation of betaAsp. The relative content of betaAsp in the peptide was successfully estimated from a unique ratio, y(l-n):y(l-n+1), corresponding to cleavages at the C- and N-termini, respectively, of the isomeric residues. The betaAsp content was consistent with measurements obtained independently by reversed phase HPLC analysis. Experiments in which neighboring amino acids adjacent to betaAsp/Asp were substituted revealed that the ratio between y(l-n) and y(l-n+1) reflected the isomerization status, while the diagnostic ion was observed only in the peptides that included an arginine residue at their C-terminus. Postsource decay experiments utilizing both the diagnostic ion and the characteristic fragment pattern could be applied to various kinds of peptides containing betaAsp.

A Comparative Study of In- and Post-Source Decays of Peptide and Preformed Ions in Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry: Effective Temperature and Matrix Effect

In-source decay (ISD) and post-source decay (PSD) of a peptide ion ([Y(6) + H](+)) and a preformed ion (benzyltriphenylphosphonium, BTPP) generated by matrix-assisted laser desorption ionization (MALDI) were investigated with time-of-flight mass spectrometry. α-Cyano-4-hydroxycinammic acid (CHCA) and 2,5-dihydroxybenzoic acid (DHB) were used as matrices. For both ions, ISD yield was unaffected by delay time, indicating rapid termination of ISD. This was taken as evidence for rapid expansion cooling of hot "early" plume formed in MALDI. CHCA was hotter than DHB for [Y(6) + H](+) while the matrix effect was insignificant for BTPP. The "early" plume temperature estimated utilizing previous kinetic results was 800-900 K, versus 400-500 K for "late" plume. The results support our previous finding that the temperature of peptide ions interrogated by tandem mass spectrometry was lower than most rough estimates of MALDI temperature.

Discrimination of Isobaric Leucine and Isoleucine Residues and Analysis of Post-Translational Modifications in Peptides by MALDI In-Source Decay Mass Spectrometry Combined with Collisional Cooling

Collisional cooling employed in an orthogonal time-of-flight mass spectrometer (o-TOF MS) stabilizes fragment ions that are generated by matrix-assisted laser desorption ionization in-source decay (MALDI ISD). By variation of the buffer gas pressure, ISD and "post-source decay" (PSD) dissociation channels can be switched on and off to some extent. Under high-pressure conditions, ISD type fragments of post-translationally modified (PTM) peptides are generated that contain even labile bound side groups; the examples of a phosphorylated and an O-glycosylated peptide are shown. At elevated laser fluences, d- and w-type fragment ions of peptides are detected as a result of high-energy side chain cleavage. This allows for differentiation of the isobaric amino acid residues leucine and isoleucine. Reduction of the cooling efficiency by lowering the buffer gas pressure results in the loss of the d-/w-type species, presumably in secondary metastable dissociation processes. This also enhances cleavage of the side groups from the PTM peptides and can be used to corroborate identification of the modification site. Furthermore, these measuring conditions generate small amounts of fragments containing sequence information about the cyclic part of a disulfide peptide by inducing symmetric and asymmetric cleavage of the intramolecular S-S bond.

Fragmentation behavior of Amadori‐peptides obtained by non‐enzymatic glycosylation of lysine residues with ADP‐ribose in tandem mass spectrometry

Mono- and poly-adenosine diphosphate (ADP)-ribosylation are common post-translational modifications incorporated by sequence-specific enzymes at, predominantly, arginine, asparagine, glutamic acid or aspartic acid residues, whereas non-enzymatic ADP-ribosylation (glycation) modifies lysine and cysteine residues. These glycated proteins and peptides (Amadori-compounds) are commonly found in organisms, but have so far not been investigated to any great degree. In this study, we have analyzed their fragmentation characteristics using different mass spectrometry (MS) techniques. In matrix-assisted laser desorption/ionization (MALDI)-MS, the ADP-ribosyl group was cleaved, almost completely, at the pyrophosphate bond by in-source decay. In contrast, this cleavage was very weak in electrospray ionization (ESI)-MS. The same fragmentation site also dominated the MALDI-PSD (post-source decay) and ESI-CID (collision-induced dissociation) mass spectra. The remaining phospho-ribosyl group (formed by the loss of adenosine monophosphate) was stable, providing a direct and reliable identification of the modification site via the b- and y-ion series. Cleavage of the ADP-ribose pyrophosphate bond under CID conditions gives access to both neutral loss (347.10 u) and precursor-ion scans (m/z 348.08), and thereby permits the identification of ADP-ribosylated peptides in complex mixtures with high sensitivity and specificity. With electron transfer dissociation (ETD), the ADP-ribosyl group was stable, providing ADP-ribosylated c- and z-ions, and thus allowing reliable sequence analyses.

Nutrition analysis by nanoparticle-assisted laser desorption/ionisation mass spectrometry

We analysed the bioactive compounds in Panax ginseng C.A. Meyer by using nanoparticle-assisted laser desorption/ionisation (nano-PALDI) mass spectrometry (MS). To this end, we prepared manganese oxide nanoparticles ( d = 5.4 nm) and developed a nano-PALDI MS method to analyse the standard ginsenosides and identify these ginsenosides in an extract of P. ginseng. The nanoparticles served as an ionisation-assisting reagent in MS. The mass spectra did not show any background interference in the low- m/z range. Our pilot study showed that the nanoparticles could ionise the standard ginsenosides and also respective lipid and ginsenosides in the extract without the aid of chemical and liquid matrices used in conventional MS methods. Analysis of the post-source decay spectra obtained using nano-PALDI MS will yield information regarding the chemical structure of the analyte.

De NovoSequencing of Tryptic Peptides Derived fromDeinococcus radioduransRibosomal Proteins Using 157 nm Photodissociation MALDI TOF/TOF Mass Spectrometry

Vacuum ultraviolet photodissociation of peptide ions in a matrix assisted laser desorption ionization (MALDI) tandem time-of-flight (TOF) mass spectrometer is used to characterize peptide mixtures derived from Deinococcus radiodurans ribosomal proteins. Tryptic peptides from 52 proteins were separated by reverse-phase liquid chromatography and spotted onto a MALDI plate. From 192 sample spots, 492 peptide ions were isolated, fragmented by both photodissociation and postsource decay (PSD), and then de novo sequenced. Three-hundred seventy-two peptides yielded sequences with 5 or more amino acids. Homology searches of these sequences against the whole bacterial proteome identified 49 ribosomal proteins, 45 of which matched with two or more peptides. Peptide de novo sequencing identified slightly more proteins than conventional database searches using Mascot and was particularly advantageous in identifying unexpected peptide modifications. In the present analysis, 52 peptide modifications were identified by de novo sequencing, most of which were not recognized by database searches.

3-Aminoquinoline Acting as Matrix and Derivatizing Agent for MALDI MS Analysis of Oligosaccharides

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a widely used method in oligosaccharide analysis. Underivatized oligosaccharides are not well-suited for that purpose due to their low ionization efficiency; however, derivatization requires tedious sample purification steps which may lead to sample losses, thereby decreasing its benefit. On-target derivatization performed by the matrix 3-aminoquinoline does not require such purification and yields Schiff bases which can be measured in positive and negative ion mode from one single spot. In negative ion mode, spectra from anionic adducts of the derivatives can be acquired from 1 fmol of oligosaccharide. Furthermore, postsource decay (PSD) fragmentation in positive and negative ion mode is enhanced, providing information on oligosaccharide sequence, linkage, and branching. Optimization of reaction conditions and matrix solution led to a complete and reproducible derivatization for all tested standard oligosaccharides. Finally, the method was applied to trifucosyllacto-N-hexaose and trifucosyl-para-lacto-N-hexaose, two isomers occurring in human breast milk samples, which were easily identified and distinguished.

Closed-Cage Tungsten Oxide Clusters in the Gas Phase

During the course of a study on the clustering of W-Se and W-S mixtures in the gas phase using laser desorption ionization (LDI) mass spectrometry, we observed several anionic W-O clusters. Three distinct species, W(6)O(19)(-), W(13)O(29)(-), and W(14)O(32)(-), stand out as intense peaks in the regular mass spectral pattern of tungsten oxide clusters suggesting unusual stabilities for them. Moreover, these clusters do not fragment in the postsource decay analysis. While trying to understand the precursor material, which produced these clusters, we found the presence of nanoscale forms of tungsten oxide. The structure and thermodynamic parameters of tungsten clusters have been explored using relativistic quantum chemical methods. Our computed results of atomization energy are consistent with the observed LDI mass spectra. The computational results suggest that the clusters observed have closed-cage structure. These distinct W(13) and W(14) clusters were observed for the first time in the gas phase.

Dissociation Kinetics of Singly Protonated Leucine Enkephalin Investigated by Time-Resolved Photodissociation Tandem Mass Spectrometry

The yields of post-source decay (PSD) and time-resolved photodissociation (PD) at 193 and 266 nm were measured for singly protonated leucine enkephalin ([YGGFL + H](+)), a benchmark in the study of peptide ion dissociation, by using tandem time-of-flight mass spectrometry. The peptide ion was generated by matrix-assisted laser desorption ionization (MALDI) using 2,5-dihydroxybenzoic acid as the matrix. The critical energy (E(0)) and entropy (DeltaS(++) at 1000 K) for the dissociation were determined by Rice-Ramsperger-Kassel-Marcus fit of the experimental data. MALDI was done for a mixture of YGGFL and Y(6) and the plume temperature determined by the kinetic analysis of [Y(6) + H](+) data were used to improve the precision of E(0) and DeltaS(++) for [YGGFL + H](+). E(0) and DeltaS(++) thus determined (E(0) = 0.67 +/- 0.08 eV, DeltaS(++) = -24.4 +/- 3.2 eu with 1 eu = 4.184 J K(-1)mol(-1)) were significantly different from those determined by blackbody infrared radiative dissociation (BIRD) (E(0) = 1.10 eV, DeltaS(++) = -14.9 eu), and by surface-induced dissociation (SID) (E(0) = 1.13 eV, DeltaS(double dagger) = -10.3 eu). Analysis of the present experimental data with the SID kinetics (and BIRD kinetics also) led to an unrealistic situation where not only PSD and PD but also MALDI-TOF signals could not be detected. As an explanation for the discrepancy, it was suggested that transition-state switching occurs from an energy bottleneck (SID/BIRD) to an entropy bottleneck (PSD/PD) as the internal energy increases.

Validation of a fast screening method for the detection of cocaine in hair by MALDI-MS

The sensitivity and specificity of a novel method of screening for cocaine in hair, based on matrix-assisted laser desorption/ionisation (MALDI) mass spectrometry (MS), have been evaluated. The method entails a rapid extraction procedure consisting of shaking 2.5 mg pulverised hair at high frequency in the presence of an acidic solution (160 microL of water, 20 microL of acetonitrile and 20 microL of 1 M trifluoroacetic acid) and a stainless-steel bullet. Following centrifugation, the supernatant is dried under a nitrogen stream, and the residue is reconstituted in 10 microL of methanol/trifluoroacetic acid (7:3; v/v). One microlitre of the extract is deposed on a MALDI sample holder previously scrubbed with graphite; an alpha-cyano-4-hydroxycinnamic acid (matrix) solution is electrosprayed over the dried sample surface to achieve a uniform distribution of matrix crystals. The identification of cocaine is obtained by post-source decay experiments performed on its MH(+) ion (m/z 304), with a limit of detection of 0.1 ng/mg of cocaine. A total of 304 hair samples were analysed in parallel by MALDI-MS and a reference gas chromatography-MS method. The obtained results demonstrate specificity and sensitivity of 100% for MALDI-MS. Evidence of cocaine presence was easily obtained even when hair samples exhibiting particularly low cocaine levels (<0.5 ng/mg) were analysed.

Peptide de Novo Sequencing Using 157 nm Photodissociation in a Tandem Time-of-Flight Mass Spectrometer

It has previously been shown that photodissociation of tryptic peptide ions with 157 nm light in a matrix-assisted laser desorption/ionization (MALDI) tandem time-of-flight (TOF) mass spectrometer generates an abundance of x-type ions. A peptide de novo sequencing algorithm has now been developed to interpret these data. By combination of photodissociation and postsource decay (PSD) spectra, the algorithm identifies x-type ions and derives peptide sequences. The confidence of amino acid assignments is evaluated by observing complementary y-, v-, and w-type ions that provide additional constraints to sequence identification. In the analysis of 31 tryptic peptides from 4 model proteins, the algorithm identified 322 (or 90.7%) of the 355 amino acids and made only 3 incorrect assignments. The other 30 amino acids were not identified because specific needed x-type ions were not detected. Based on the observation of v- and w-type ions, 45 of 50 detected leucine and isoleucine residues were successfully distinguished and there was only one mistake. The remaining four residues were not distinguished because the corresponding v- and w-type ions were not detected. These de novo sequencing results translated into successful identification of proteins through homology searches. To evaluate the robustness of the present sequencing approach, a collection of 266 tryptic peptides from 23 model proteins were analyzed and then sequenced. A total of 167 peptides yielded sequence tags of 5 or more residues. In 5 peptides, 1 or 2 residues were incorrectly assigned.

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry analysis of oligosaccharides and oligosaccharide alditols obtained by hydrolysis of agaroses and carrageenans, two important types of red seaweed polysaccharides

MALDI-TOF mass spectrometry analyses of several oligosaccharides (aldoses) and oligosaccharide alditols derived from agaroses, kappa- and iota-carrageenans using different matrices (2,5-dihydroxybenzoic acid, nor-harmane, ferulic acid, and the ionic liquid matrices 2,5-dihydroxybenzoic acid–n-butylamine and ferulic acid–n-butylamine) were conducted. These carbohydrates were selected as model compounds to study the MALDI prompt and post-source decay (PSD) fragmentation processes of both families of oligosaccharides. Sulfated alditols showed in the negative-ion mode the molecular ion as [M−Na]− together with the species yielded by their prompt fragmentation (mainly desulfation) while the sulfated oligosaccharides (aldoses) showed mainly glycosidic prompt fragmentation (glycosidic C-cleavages and desulfation). Non-sulfated aldoses and alditols, which could only be analyzed in positive-ion mode ([M+Na]+), did not suffer any prompt fragmentation. The former yielded cross-ring fragmentation in the PSD mode. Best results were obtained by using 2,5-dihydroxybenzoic acid and/or nor-harmane as matrices for all the compounds studied.

Influence of basic residues on dissociation kinetics and dynamics of singly protonated peptides: time‐resolved photodissociation study

Product ion yields in postsource decay and time-resolved photodissociation at 193 and 266 nm were measured for some peptide ions with lysine ([KF6 + H]+, [F6K + H]+, and [F3KF3 + H]+) formed by matrix-assisted laser desorption ionization. The critical energy (E0) and entropy (DeltaS(double dagger)) were determined by RRKM fitting of the data. The results were similar to those found previously for peptide ions with histidine. To summarize, the presence of a basic residue, histidine or lysine, inside a peptide ion retarded its dissociation by lowering DeltaS(double dagger). On the basis of highly negative DeltaS(double dagger), presence of intramolecular interaction involving a basic group in the transition structure was proposed.

Analysis of human plasma lipids and soybean lecithin by means of high‐performance thin‐layer chromatography and matrix‐assisted laser desorption/ionization mass spectrometry

An improved analytical strategy for the analysis of complex lipid mixtures using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) in combination with high-performance thin-layer chromatography (HPTLC) is reported. Positive ion MALDI RTOF MS was applied as a rapid screening tool for the various neutral (e.g. triacylglycerols) and polar (e.g. glycerophospholipids and -sphingolipids) lipid classes derived from crude lipid extracts of e.g. human plasma as well as soybean lecithin. Finally, MALDI seamless post-source decay (PSD) product ion analysis was performed in order to obtain further structural information (head- and acyl-group identification) of selected lipid species and structure verification. A Coomassie Brilliant Blue R-250 staining protocol for lipids on HPTLC plates was evaluated and was found to be fully compatible with subsequent MALDI-MS. Lipids were analyzed after elution from the HPTLC phase material of the selected band (corresponding to certain lipid classes) by using the proper organic solvent mixture or in few cases directly from the HPTLC plates (a type of on-line HPTLC/MALDI-MS coupling). More than 70 distinct lipid species from seven different lipid classes in the range between m/z 500 and 1500 could be identified from the lipid extracts of human plasma and soybean lecithin, respectively. The general high sensitivity of MALDI-MS detection allowed the analysis of even minor lipid classes from only very small volumes of human plasma (50 microL). The combination of HPTLC, Coomassie staining and positive ion MALDI curved field RTOF-MS represents a straightforward strategy during lipidomics studies of food and clinically relevant human lipid samples.

Time-Resolved Photodissociation Study of Singly Protonated Peptides with a Histidine Residue Generated by Matrix-Assisted Laser Desorption Ionization: Dissociation Rate Constant and Internal Temperature

Product ion yields in post-source decay and time-resolved photodissociation at 193 and 266 nm were measured for some peptide ions with a histidine residue ([HF(6) + H](+), [F(6)H + H](+), and [F(3)HF(3) + H](+)) formed by matrix-assisted laser desorption ionization (MALDI). Compared with similar data for peptide ions without any basic residue reported previously, significant reduction in dissociation efficiency was observed. Internal temperatures (T) of the peptide ions and their dissociation kinetic parameters-the critical energy (E(0)) and entropy (DeltaS(double dagger))-were determined by the method reported previously. Slight decreases in E(0), DeltaS(double dagger), and T were responsible for the histidine effect-reduction in dissociation rate constant. Regardless of the presence of the residue, DeltaS(double dagger) was far more negative than previous quantum chemical results. Based on this, we propose the existence of transition structures in which the nitrogen atoms in the histidine residue or at the N-terminus coordinate to the reaction centers. Reduction in T in the presence of a histidine residue could not be explained based on popular models for ion formation in MALDI, such as the gas-phase proton transfer model.

Alterations in Fatty Acid Utilization and an Impaired Antioxidant Defense Mechanism Are Early Events in Podocyte Injury: A Proteomic Analysis

Ultrastructural alterations of podocytes are closely associated with loss of glomerular filtration function. In the present study, we explored changes at the proteome level that paralleled the disturbances of podocyte architecture in the early stages of puromycin aminonucleoside (PA) nephrosis in vivo. Using two-dimensional fluorescence difference gel electrophoresis and vacuum matrix-assisted laser desorption/ionization mass spectrometry combined with postsource decay fragment ion analysis and high-energy collision-induced dissociation tandem mass spectrometry, 23 differentially expressed protein spots, corresponding to 16 glomerular proteins that are involved in various cellular functions, were unambiguously identified, and a subset was corroborated by Western blot analysis. The majority of these proteins were primarily related to fatty acid metabolism and redox regulation. Key enzymes of the mitochondrial beta-oxidation pathway and antioxidant enzymes were consistently down-regulated in PA nephrosis. These changes were paralleled by increased expression levels of CD36. PA treatment of murine podocytes in culture resembled these specific protein changes in vitro. In this cell system, the modulatory effects of albumin-bound fatty acids on the expression levels of Mn-superoxide dismutase in response to PA were demonstrated as well. Taken together, these results indicate that a disrupted fatty acid metabolism in concert with an impaired antioxidant defense mechanism in podocytes may play a role in the early stages of PA-induced lesions in podocytes.

Utility of mass spectrometry for proteome analysis: part II. Ion-activation methods, statistics, bioinformatics and annotation

This is the second article in a series, intended as a tutorial to provide the interested reader with an overview of the concepts not covered in part I, such as: the principles of ion-activation methods, the ability of mass-spectrometric methods to interface with various proteomic strategies, analysis techniques, bioinformatics and data interpretation and annotation. Although these are different topics, it is important that a reader has a basic and collective understanding of all of them for an overall appreciation of how to carry out and analyze a proteomic experiment. Different ion-activation methods for MS/MS, such as collision-induced dissociation (including postsource decay) and surface-induced dissociation, electron capture and electron-transfer dissociation, infrared multiphoton and blackbody infrared radiative dissociation have been discussed since they are used in proteomic research. The high dimensionality of data generated from proteomic studies requires an understanding of the underlying analytical procedures used to obtain these data, as well as the development of improved bioinformatics tools and data-mining approaches for efficient and accurate statistical analyses of biological samples from healthy and diseased individuals, in addition to determining the utility of the interpreted data. Currently available strategies for the analysis of the proteome by mass spectrometry, such as those employed for the analysis of substantially purified proteins and complex peptide mixtures, as well as hypothesis-driven strategies, have been elaborated upon. Processing steps prior to the analysis of mass spectrometry data, statistics and the several informatics steps currently used for the analysis of shotgun proteomic experiments, as well as proteomics ontology, are also discussed.

Effect of Gas Pressure and Gas Type on the Fragmentation of Peptide and Oligosaccharide Ions Generated in an Elevated Pressure UV/IR-MALDI Ion Source Coupled to an Orthogonal Time-of-Flight Mass Spectrometer

Matrix-assisted laser desorption ionization (MALDI) allows for the mass spectrometric (MS) analysis of thermally labile, non-volatile biomolecules. However, some residual analyte fragmentation typically accompanies the phase transition from the condensed to the gas phase and following plume expansion, even under optimized conditions. In-source decay (ISD) and post-source decay (PSD) MALDI MS are two techniques that make use of these phenomena and that can provide useful structural information by producing characteristic fragment ions of the analyte compounds. In orthogonal extracting time-of-flight mass spectrometry (o-TOF-MS), the pressure of the cooling gas in the ion source has a strong influence on the extent of analyte ion fragmentation. We investigated the effect of this parameter on peptide and oligosaccharide fragmentation by examining a range of pressures (from 0.05-1.8 mbar) in combination with seven different buffer gases (He, Ne, Ar, N(2), CO(2), CH(3), isobutane). Ions were generated by ultraviolet (UV) and/or by infrared (IR) MALDI. The influence of the ion extraction voltage on the analyte fragmentation also was investigated for a selected set of gas parameters. We observed that individual fragment ions exhibit characteristic fragment yield-pressure dependencies that can be classified into three groups. Type I ions resemble species that are also found in MALDI PSD MS analysis, while type II ions resemble typical ISD fragments. The yield-pressure relationship of type III ions suggests that these are the result of a combination of both processes. Comparing the yields of fragmentation for the different buffer gases reveals a correlation between their internal degrees of freedom and their collisional cooling efficiency. Changing the buffer gas pressure and/or extraction field provides an easy means to influence analyte ion fragmentation and to switch from the primary production of one type of fragment species to another. The method can therefore facilitate the structural characterization of MALDI-generated ions.