Electrospray tandem mass spectrometry of intact beta-chain hemoglobin variants

In this report, we present data to illustrate how human hemoglobin (Hb) variants can be identified by electrospray tandem mass spectrometry (MS/MS) of the intact Hb chains following the one-step dilution of whole blood. MS/MS spectra were recorded on a series of intact beta-chain human Hb variants. The resultant spectra were interpreted, and using the information gleaned from the fragmentation patterns of known variants, two unknown beta-chain variants were characterized solely by this mass spectrometric method. Fragment ions that serve to identify beta-chain variants were identified. The fragmentation patterns of the intact beta-chain [M + 18H]18+ ions showed classical facile cleavages adjacent to acidic residues and N-terminal to proline residues, with Thr50-Pro51 being the most prominent cleavage site. Abundant product ions were formed by peptide bond cleavage in the regions close to the termini of the beta chain, the central region being less well-represented in the MS/MS spectra. Nearly 50% of the beta-chain primary structure could be determined by MS/MS of the intact chain. However, analysis of the Hb variants where mutations have occurred in the inner region (residues 58-111) of the beta globin proved to be difficult and required mass spectrometric analysis of their tryptic peptides for a complete identification.     

A statistical model for identifying proteins by tandem mass spectrometry

A statistical model is presented for computing probabilities that proteins are present in a sample on the basis of peptides assigned to tandem mass (MS/MS) spectra acquired from a proteolytic digest of the sample. Peptides that correspond to more than a single protein in the sequence database are apportioned among all corresponding proteins, and a minimal protein list sufficient to account for the observed peptide assignments is derived using the expectation-maximization algorithm. Using peptide assignments to spectra generated from a sample of 18 purified proteins, as well as complex H. influenzae and Halobacterium samples, the model is shown to produce probabilities that are accurate and have high power to discriminate correct from incorrect protein identifications. This method allows filtering of large-scale proteomics data sets with predictable sensitivity and false positive identification error rates. Fast, consistent, and transparent, it provides a standard for publishing large-scale protein identification data sets in the literature and for comparing the results obtained from different experiments.     

Identification of N-linked glycosylation sites using glycoprotein digestion with pronase prior to MALDI tandem time-of-flight mass spectrometry

Glycopeptides are typically prepared by cleaving the proteins with specific proteolytic enzymes, such as trypsin. The resulting glycopeptides tend to have weak mass spectrometry ion signals (ESI or MALDI) due to their relatively large molecular weight. The identification of glycosylation sites with tandem mass spectrometry is further complicated by fragmentation of both the peptide backbone and the glycan moiety. We explored a method using a nonspecific enzyme, pronase, to generate small glycopeptides (between two and six amino acids). These glycopeptides were enriched and desalted using a microscale hydrophilic interaction chromatography extraction device prior to MALDI QTof MS analysis. MALDI matrix, 2, 5-dihydroxybenzoic acid, doped with ammonium triscitrate, was utilized for analysis. Sodiated ions were observed as minor ions, while protonated ions were enhanced dramatically with this matrix. Collision-induced dissociation was performed on both the protonated and sodiated ions. MS/MS fragmentation spectra reveal that proton has greater affinity for the peptide moiety, while the sodium cation tends to associate with the sugar moiety. Characteristic fragment patterns allowed for identifications of glycosylation sites for both the protonated and the sodiated precursor ions. Model proteins, horseradish peroxidase and alpha1-acid glycoproteins, were analyzed to illustrate the identification of N-linked glycosylation sites and data interpretation algorithm.     

Direct analysis of bacterial fatty acids by Curie-point pyrolysis tandem mass spectrometry

Although pyrolysis-mass spectrometry (Py-MS) has been used for bacterial taxonomy, many of the mass spectral peaks used for discrimination of organisms have not been correlated to known biomolecules. This work presents the discrimination of five bacterial species based on Py-MS patterns containing only peaks that can be correlated to fatty acids and fatty acid derivatives. These correlations were confirmed by pyrolysis-tandem mass spectrometry of authentic standards and the organisms. The pattern recognition procedures used gave better results when only the fatty acid peaks were used in the analysis than when full spectra were used.     

Processing complex mixtures of intact proteins for direct analysis by mass spectrometry

For analysis of intact proteins by mass spectrometry (MS), a new twist to a two-dimensional approach to proteome fractionation employs an acid-labile detergent instead of sodium dodecyl sulfate during continuous-elution gel electrophoresis. Use of this acid-labile surfactant (ALS) facilitates subsequent reversed-phase liquid chromatography (RPLC) for a net two-dimensional fractionation illustrated by transforming thousands of intact proteins from Saccharomyces cerevisiae to mixtures of 5-20 components (all within approximately 5 kDa of one another) for presentation via electrospray ionization (ESI) to a Fourier transform MS (FTMS). Between 3 and 13 proteins have been detected directly using ESI-FTMS (or MALDI-TOF), and the fractionation showed a peak capacity of approximately 400 between 0 and 70 kDa. A probability-based identification was made automatically from raw MS/MS data (obtained using a quadrupole-FTMS hybrid instrument) for one protein that differed from that predicted in a yeast database of approximately 19,000 protein forms. This ALS-PAGE/RPLC approach to proteome processing ameliorates the "front end" problem that accompanies direct analysis of whole proteins and assists the future realization of protein identification with 100% sequence coverage in a high-throughput format.     

Time-condensed analyses by mass spectrometry

Research has focused on the development of a new set of mathematical algorithms, encoded in C(++), when combined with a thermal desorption sample introduction system provides quantitative analysis of a wide mixture of organic compounds in under 10 min by gas chromatography/mass spectrometry. The overall goal is to condense the time of analysis, including both the times required for sample preparation and for chromatographic separation. In this paper, results are presented where compound identification has been made for polychlorinated biphenyls, chlorinated pesticides, and polycyclic aromatic hydrocarbons present in the same solution and where gas chromatography separation times have been reduced from 40 to 5 min. For the latter, all compounds elute within 3.5 min, with structural isomers identified as the same compound. The 5-min analysis provides the foundation for rapid screening and on-line chemical measurements of multicomponent mixtures. Results are also presented where these same compounds are quantitatively analyzed in 10 min, with structural isomers identified individually, in the presence of a (25% v/v) weathered gasoline/engine oil mixture. Time-condensed complex mixture detection is now feasible making possible quantitative, high-throughput sample analyses.     

Identification of microbial mixtures by capillary electrophoresis/selective tandem mass spectrometry

In this paper, we propose a new strategy for identifying specific bacteria in bacterial mixtures by using CE-selective MS/MS of peptide marker ions associated with the bacteria of interest. We searched the CE-MS/MS spectra acquired from the proteolytic digests of pure bacterial cell extracts against protein databases. The identified peptides that match the protein associated with the corresponding species were selected as marker ions for bacterial identification. Specific peptide marker ions were obtained for each of the following three pathogens: Pseudomonas aeruginasa, Staphylococcus aureus, and Staphylococcus epidermidis. To identify a bacterial species in a sample, we performed CE-MS/MS analysis of the selected marker ions in the proteolytic digest of the cell extract and then performed protein database searches. The selected peptides that we identified correctly from Xcorr values ranking at the top of the search results allowed us to identify the corresponding bacterial species present in the sample. We have applied this method successfully to the identification of various mixtures of the three pathogens. Even minor bacterial species present at a concentration of 1% can be identified with great confidence. This method for CE-MS/MS analysis of bacteria-specific marker peptides provides excellent selectivity and high accuracy when identifying bacterial species in complex systems. In addition, we have used this approach to identify P. aeruginasa in a saliva sample spiked with E.coli and P. aeruginasa.     

Identification of proteins in complexes by solid-phase microextraction/multistep elution/capillary electrophoresis/tandem mass spectrometry.

A method to directly identify proteins in complex mixtures by solid-phase microextraction (micro-SPE)/multistep elution/capillary electrophoresis (CE)/tandem mass spectrometry (MS/MS) is described. A sheathless liquid-metal junction interface is used to interface CE and electrospray ionization MS/MS. A subfemtomole detection limit is achieved for protein identification through database searching using MS/MS data. The SPE serves as a semiseparation dimension using an organic-phase step-elution gradient in combination with the second separation dimension for increased resolving power of complex peptide mixtures. This approach improves the concentration detection limit for CE and allows more proteins in complex mixtures to be identified. A 75-protein complex from yeast ribosome is analyzed using this method and 80-90% of the proteins in the complex can be identified by searching the database using the MS/MS data from a complete analysis. This multidimensional CE/MS/MS methodology provides an alternative to multidimensional liquid chromatography/MS/MS for direct identification of small amounts of protein in mixtures.     

Identification of in-gel digested proteins by complementary peptide mass fingerprinting and tandem mass spectrometry data obtained on an electrospray ionization quadrupole time-of-flight mass spectrometer

The present study reports a procedure developed for the identification of SDS-polyacrylamide gel electrophoretically separated proteins using an electrospray ionization quadrupole time-of-flight mass spectrometer (Q-TOF MS) equipped with pressurized sample introduction. It is based on in-gel digestion of the proteins without previous reduction/alkylation and on the capability of the Q-TOF MS to provide data suitable for peptide mass fingerprinting database searches and for tandem mass spectrometry (MS/MS) database searches (sequence tags). Omitting the reduction/alkylation step reduces sample contamination and sample loss, resulting in increased sensitivity. Omitting this step can leave disulfide-connected peptides in the analyte that can lead to misleading or ambiguous results from the peptide mass fingerprinting database search. This uncertainty, however, is overcome by MS/MS analysis of the peptides. Furthermore, the two complementary MS approaches increase the accuracy of the assignment of the unknown protein. This procedure is thus, highly sensitive, accurate, and rapid. In combination with pressurized nanospray sample introduction, it is suitable for automated sample handling. Here, we apply this approach to identify protein contaminants observed during the purification of the yeast DNA mismatch repair protein Mlh 1.     

Distinguishing and quantifying peptides and proteins containing D-amino acids by tandem mass spectrometry

Tandem mass spectrometry (MS/MS) utilizing both electron capture dissociation (ECD) and collisionally activated dissociation (CAD) was used to develop a qualitative and quantitative analytical method for chiral analysis of individual amino acid residues in polypeptides. ECD produced a more distinct chiral recognition than CAD, which is attributed to the smaller degree of vibrational excitation in ECD. Several peptide and protein model systems were used in this study, including the smallest known protein, tryptophan cage, a lactoferrin peptide, and the biologically relevant opioid peptide, dermorphin. An adaptation of the kinetic method was used to quantify the degree of separation between fragmentation patterns of stereoisomeric peptides as a function of fragment ion abundances. The obtained calibration scale for relative abundances of d-amino acids in diastereomeric peptide mixtures was accurate to 1% for ECD and to 3-5% for CAD. It was found that separation and quantification of stereoisomers could be advantageously performed by nanoflow reversed-phase liquid chromatography, with the objective of on-line MS/MS limited to stereoisomer identification. This technique shows promise for the analysis of chiral substitution in peptides and proteins, broadening the application area for tandem mass spectrometry.     

Identification of trichloroethanol visualized proteins from two-dimensional polyacrylamide gels by mass spectrometry

Proteins visualized by 2,2,2-trichloroethanol (TCE) on two-dimensional electrophoresis gels are efficiently identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and MS/MS. In a previous study, a method was developed that placed TCE in the polyacrylamide gel so that protein bands can be visualized without staining in less than 5 min. A visible fluorophore is generated by reaction of TCE with tryptophan that allows for protein visualization. In this study, MALDI-TOF MS and LC-MS/MS are used to identify randomly selected Escherichia coli proteins. The identification of TCE visualized proteins is compared to the identification of Coomassie brilliant blue (CBB) stained proteins from two-dimensional gel electrophoresis of E. coli proteins. This study demonstrated that TCE visualized proteins are compatible with protein identification by MALDI-TOF peptide mass fingerprinting. For 10 randomly selected spots, TCE visualization lead to statistically significant identification of 5 proteins and CBB visualization lead to identification of 6 proteins. TCE visualized proteins are also shown to be well suited for protein identification using LC-MS/MS. In 16 spots selected for MS/MS analysis, TCE samples lead to the identification of 79 peptides; while CBB samples lead to the identification of 65 peptides. TCE samples also supported the identification of more proteins. The low stoichiometry of labeling of tryptophan residues does not require inclusion of this modification for database searches. In addition to being a rapid visualization technique compatible with MS, TCE visualization utilizes rapid washing conditions for sample preparation of proteins spots excised from polyacrylamide gels.     

Bidimensional tandem mass spectrometry for selective identification of nitration sites in proteins

Nitration of protein tyrosine residues is very often regarded as a molecular signal of peroxynitrite formation during development, oxidative stress, and aging. However, protein nitration might also have biological functions comparable to protein phosphorylation, mainly in redox signaling and in signal transduction. The major challenge in the proteomic analysis of nitroproteins is the need to discriminate modified proteins, usually occurring at substoichiometric levels from the large amount of nonmodified proteins. Moreover, precise localization of the nitration site is often required to fully describe the biological process. Existing methodologies essentially rely on immunochemical techniques either using 2D-PAGE fractionation in combination with western blot analyses or exploiting immunoaffinity procedures to selectively capture nitrated proteins. Here we report a totally new approach involving dansyl chloride labeling of the nitration sites that rely on the enormous potential of MSn analysis. The tryptic digest from the entire protein mixture is directly analyzed by MS on a linear ion trap mass spectrometer. Discrimination between nitro- and unmodified peptide is based on two selectivity criteria obtained by combining a precursor ion scan and an MS3 analysis. This new procedure was successfully applied to the identification of 3-nitrotyrosine residues in complex protein mixtures.     

Imaging and 3D elemental characterization of intact bacterial spores by high-resolution secondary ion mass spectrometry

We present a quantitative, imaging technique based on nanometer-scale secondary ion mass spectrometry for mapping the 3D elemental distribution present in an individual micrometer-sized Bacillus spore. We use depth profile analysis to access the 3D compositional information of an intact spore without the additional sample preparation steps (fixation, embedding, and sectioning) typically used to access substructural information in biological samples. The method is designed to ensure sample integrity for forensic characterization of Bacillus spores. The minimal sample preparation/alteration required in this methodology helps to preserve sample integrity. Furthermore, the technique affords elemental distribution information at the individual spore level with nanometer-scale spatial resolution and high (microg/g) analytical sensitivity. We use the technique to map the 3D elemental distribution present within Bacillus thuringiensis israelensis spores.     

Affinity chromatographic selection of carbonylated proteins followed by identification of oxidation sites using tandem mass spectrometry

It has been shown that oxidatively modified forms of proteins accumulate during oxidative stress, aging, and in some age-related diseases. One of the unique features of a wide variety of routes by which proteins are oxidized is the generation of carbonyl groups. This paper reports a method for the isolation of oxidized proteins, which involves (1) biotinylation of oxidized proteins with biotin hydrazide and (2) affinity enrichment using monomeric avidin affinity chromatography columns. The selectivity of the method was validated by adding in vitro oxidized biotinylated BSA to a yeast lysate and showing that the predominant protein recovered was BSA. This method was applied to the question of whether large doses of 2-nitropropane produce oxidized proteins. A study of rat liver homogenates showed that animals dosed with 2-nitropropane produced 17 times more oxidized protein than controls in 6 h. Tryptic digestion of these oxidized proteins followed by reversed-phase chromatography and tandem mass spectrometry led to the identification of 14 peptides and their parent proteins. Nine of the 14 identified peptides were found to carry 1 or 2 oxidation sites and 5 of the 9 peptides were biotinylated. The significance of this affinity method is that it allows the isolation of oxidized proteins from the rest of the proteome and facilitates their identification. In some cases, it is even possible to identify the site of oxidation.     

Automated identification of amino acid sequence variations in proteins by HPLC/microspray tandem mass spectrometry

Amino acid sequence variations resulting from single-nucleotide polymorphisms (SNPs) were identified using a novel mass spectrometric method. This method obtains 99+% protein sequence coverage for human hemoglobin in a single LC-microspray tandem mass spectrometry (microLC-MS/MS) experiment. Tandem mass spectrometry data was analyzed using a modified version of the computer program SEQUEST to identify the sequence variations. Conditions of sample preparation, chromatographic separation, and data collection were optimized to correctly identify amino acid changes in six variants of human hemoglobin (Hb C, Hb E, Hb D-Los Angeles, Hb G-Philadelphia, Hb Hope, and Hb S). Hemoglobin proteins were isolated and purified, dehemed, (S)-carboxyami-domethylated, and then subjected to a combination proteolytic digestion to obtain a complex peptide mixture with multiple overlaps in sequence. Reversed-phase chromatographic separation of peptides was achieved on-line with MS utilizing a robust fritless microelectrospray interface. Tandem mass spectrometry was performed on an ion trap mass spectrometer using automated data-dependent MS/MS procedures. Tandem mass spectra were collected from the five most abundant ions in each scan using dynamic and isotopic exclusion to minimize redundancy. The spectra were analyzed by a version of the SEQUEST algorithm modified to identify amino acid substations resulting from SNPs.     

Identification of anomeric configuration of underivatized reducing glucopyranosyl-glucose disaccharides by tandem mass spectrometry and multivariate analysis

The possibility of discrimination of the anomeric configuration (alpha or beta) of underivatized reducing glucopyranosyl-glucose disaccharides, using a hybrid mass spectrometer Q-TOF 2 (Micromass), a linear ion trap LXQ (Thermo), and a triple quadrupole Quattro (Micromass) with an electrospray source (ESI) was investigated. Differences observed in the relative abundances of specific product ions obtained from collisionally induced dissociation of the [M + Li]+ adducts were statistically analyzed, and discriminant analysis was performed. MANOVA has shown that anomeric configuration has influence on the combined dependent variables (relative abundances of m/z product ions) in all the three mass spectrometers used (Q-TOF 2, LIT, and QqQ). Discriminant analysis has shown that, in all instruments, it is possible to discriminate anomeric configurations and to build a diagnostic model. These diagnostic differences are even more relevant considering that no derivatization procedures are needed for obtaining this structural information. The Q-TOF 2 instrument has been shown to give data that allowed us to build a model with better discriminant power (Wilks' lambda value of 0.014) followed by the QqQ instrument (Wilks' lambda value of 0.029) and the LIT instrument (Wilks' lambda value of 0.037).     

On-line hollow-fiber flow field-flow fractionation-electrospray ionization/time-of-flight mass spectrometry of intact proteins

Capabilities of mass spectrometry for the analysis of intact proteins can be increased through separation methods. Flow field-flow fractionation (FlFFF) is characterized by the particularly "soft" separation mechanism, which is ideally suited to maintain the native structure of intact proteins. This work describes the original on-line coupling between hollow-fiber FlFFF (HF FlFFF), the microcolumn variant of FlFFF, and electrospray ionization/time-of-flight mass spectrometry (ESI/TOFMS) for the analysis and characterization of intact proteins. The results show that the native (or pseudonative) structure of horse heart myoglobin and horseradish peroxidase is maintained. Sample desalting is also observed for horse heart myoglobin. Correlation between the molar mass values independently measured by HF FlFFF retention and ESI/TOFMS allows us to confirm the protein aggregation features of bovine serum albumin and to indicate possible changes in the quaternary structure of human hemoglobin.     

Rapid screening for taxanes by tandem mass spectrometry.

A highly specific and sensitive method is described for determining taxol, cephalomannine, and baccatin III in crude plant extracts. Radical anions of the taxanes are formed by desorption chemical ionization, and a parent tandem mass spectrometric scan is used to recognize these compounds by their characteristic dissociations. The limit of detection of the individual taxanes in typical plant matrices is less than 500 pg when all three species are screened simultaneously. Because of the sensitivity of the method, extraction times can be shortened to 30 min and crude extracts can be examined at the rate of 6/h. Detection of all three taxanes extracted from a single Taxus cuspidata needle in a combined extraction/analysis time of less than 1 h is demonstrated.     

Identification of rotenone-induced modifications in alpha-synuclein using affinity pull-down and tandem mass spectrometry

Parkinson's disease is a movement disorder that results from a loss of dopaminergic neurons in the substantia nigra. The disease is characterized by mitochondrial dysfunction, oxidative stress, and the presence of "Lewy body" inclusions enriched with aggregated forms of alpha-synuclein, a presynaptic protein. Although alpha-synuclein is modified at various sites in Lewy bodies, it is unclear how sequence-specific posttranslational modifications modulate the aggregation of the protein in oxidatively stressed neurons. To begin to address this problem, we developed an affinity pull-down/mass spectrometry method to characterize the primary structure of histidine-tagged alpha-synuclein isolated from catecholaminergic neurons. Using this method, we mapped posttranslational modifications of alpha-synuclein from untreated neurons and neurons exposed to rotenone, an inhibitor of mitochondrial complex I. Various posttranslational modifications suggestive of oxidative damage or repair were identified in a region comprising a 20-residue stretch in the C-terminal part of the protein. The results indicate that alpha-synuclein is subject to discrete posttranslational modifications in neurons with impaired mitochondrial function. Our affinity pull-down/mass spectrometry method is a useful tool to examine how specific modifications of alpha-synuclein contribute to neurologic disorders such as Parkinson's disease.     

Mass spectrometry and tandem mass spectrometry of citrus limonoids

Methods for atmospheric pressure chemical ionization tandem mass spectrometry (APCI-MS/MS) of citrus limonoid aglycones and electrospray ionization tandem mass spectrometry (ESI-MS/MS) of limonoid glucosides are reported. The fragmentation patterns of four citrus limonoid aglycones (limonin, nomilin, obacunone, and deacetylnomilin) and six limonoid glucosides, that is, limonin 17-beta-D-glucopyranoside (LG), nomilin 17-beta-D-glucopyranoside (NG), nomilinic acid 17-beta-D-glucopyranoside (NAG), deacetyl nomilinic acid 17-beta-D-glucopyranoside (DNAG), obacunone 17-beta-D-glucopyranoside (OG), and obacunoic acid 17-beta-D-glucopyranoside (OAG) were investigated using a quadruple mass spectrometer in low-energy collisionally activated dissociation (CAD). The four limonoid aglycones and four limonoid glucosides (LG, OG, NAG, and DNAG) were purified from citrus seeds; the other two limonoid glucosides (NG and OAG) were tentatively identified in the crude extract of grapefruit seeds by ESI mass spectrometry in both positive and negative ion analysis. Ammonium hydroxide or acetic acid was added to the mobile phase to facilitate ionization. During positive ion APCI analysis of limonoid aglycones, protonated molecular ion, [M + H]+, or adduct ion, [M + NH3 + H]-, was formed as base peaks when ammonium hydroxide was added to the mobile phase. Molecular anions or adduct ions with acetic acid ([M + HOAc - H] and [M + HOAc]-) or a deprotonated molecular ion were produced during negative ion APCI analysis of limonoid aglycones, depending on the mobile-phase modifier used. Positive ion ESI-MS of limonoid glucosides produced adduct ions of [M + H + NH3]+, [M + Na]+, and [M + K]+ when ammonium hydroxide was added to the mobile phase. After collisionally activated dissociation (CAD) of the limonoid aglycone molecular ions in negative ion APCI analysis, fragment ions indicated structural information of the precursor ions, showing the presence of methyl, carboxyl, and oxygenated ring structure. CAD of the adduct ion [M + H + NH3]+ of limonoid glucosides produced the aglycone moiety corresponding to each glucoside. The combination of mass spectrometry and tandem mass spectrometry provides a powerful technique for identification and characterization of citrus limonoids.