Identification of selenium-containing glutathione S-conjugates in a yeast extract by two-dimensional liquid chromatography with inductively coupled plasma MS and nanoelectrospray MS/MS detection

An approach for the identification of unknown selenium-containing biomolecules was developed, enabling the identification of selenodiglutathione (GS-Se-SG) and the mixed selenotrisulfide of glutathione and cysteinylglycine (GS-Se-SCG) in aqueous yeast extracts. The method consists of two-dimensional liquid chromatography, inductively coupled plasma mass spectrometry (ICPMS) and nanoelectrospray tandem mass spectrometry. Analytes were separated by size-exclusion chromatography followed by preconcentration and separation on a porous graphitic carbon HPLC column. The HPLC effluent was monitored for selenium by ICPMS, and two selenium-containing fractions were isolated and analyzed by nanoelectrospray MS. The nanoelectrospray technique has a low sample consumption of approximately 80 nL/min, enabling a preconcentration of the sample to a few microliters. Mass spectra of the two fractions showed the characteristic Se isotopic pattern centered at m/z 693.1 and 564.0 for the [M + H]+ 80Se ions. MS/MS spectra of adjacent parent ions confirmed the presence of Se. The two selenium species were identified as GS-Se-SG and GS-Se-SCG by collision induced dissociation (CID). The accurately measured masses of the most abundant 691 and 693 u parent ions are in good agreement (differences = 3 ppm) with the theoretical masses. To our knowledge, this is the first identification of GS-Se-SG and GS-Se-SCG in biological matrixes by MS/MS.     

Analysis of epipolythiodioxopiperazines in fungus Chaetomium cochliodes using HPLC-ESI-MS/MS/MS

A series of epipolythiodioxopiperazines in the fungus Chaetomium cochliodes was investigated using reversed-phase liquid chromatography with diode array detection and electrospray quadrupole time-of-flight-type tandem mass spectrometry in the positive ion mode. The fragmentation of protonated molecular ions including low-abundance parent ions, [M+H]+ for five known epipolythiodioxopiperazines, dethiotetra(methylthio)chetomin, chaetocochins A-C, and chetomin, was carried out using low-energy collision-induced electrospray ionization tandem spectrometry. It was found that McLafferty rearrangements occurred in the CID processes and produced a complementary pair of characteristic fragment ions containing piperazine rings (fused and unfused), especially to determine the number of S atoms on each ring. The fragmentation differential between [M+H]+ and [M+Na]+ was uncovered. Complementary fragmentation information obtained from [M+H]+ and [M+Na]+ precursor ions is especially valuable for rapid identification of epipolythiodioxopiperazines. A likely known compound, possibly related to chetoseminudin A, and three new species of epipolythiodioxopiperazines from the fungus C. cochliodes were identified or tentatively characterized based on tandem mass spectra of known ones.     

Molecular weight distributions of heavy aromatic petroleum fractions by Ag+ electrospray ionization mass spectrometry

The ability of electrospray ionization mass spectrometry (ESI MS) to analyze heavy aromatic petroleum fractions using silver nitrate as a reagent compound to form characteristic adduct ions has been examined. The complexation of aromatic compounds containing long alkyl substituents with the silver ion leads to the formation of abundant adduct ions such as [M + Ag]+ and [2M + Ag]+. The concentration of the [2M + Ag]+ ions can be reduced by increasing the sampling cone voltage. Molecular ions and other adduct ions may also be formed depending on the structure of the aromatic molecule. Results obtained from the analysis of representative heavy petroleum fractions and vacuum residues by the Ag+ ESI MS method and conventional ionization methods were in good agreement. The current method extends the applicability of electrospray ionization to the analysis of neutral hydrocarbons in heavy aromatic petroleum fractions. It is simple and compatible with widely available LC/MS instrumentation. The extreme complexity of the Ag     

Parent and neutral loss monitoring on a quadrupole ion trap mass spectrometer: screening of acylcarnitines in complex mixtures

A novel and practical technique for performing both parent and neutral loss (P&NL) monitoring experiments on a quadrupole ion trap mass spectrometer is presented. This technique is capable of performing scans analogous to the parent and neutral loss scans routinely applied on tandem-in-space instruments and allows for the screening of a sample to detect analytes of a specific compound class on a chromatographic time-scale. Acylcarnitines were chosen as the model compound class to demonstrate the analytical utility of P&NL monitoring because of their amenability to electrospray ionization (ESI), their unique and informative MS/MS fragmentation pattern, and their importance in biological functions. The [M + H]+ ions of all acylcarnitines dissociate to produce neutral losses of 59 and 161 amu and common product ions at m/z 60, 85, and 144. Both the neutral loss monitoring of 59 amu and the parent ion monitoring of m/z 85 are shown to be capable of identifying acylcarnitine [M + H]+ ions in a synthetic mixture and spiked pig plasma. The neutral loss monitoring of 59 amu is successful in detecting acylcarnitines in an unspiked pig plasma sample.     

Characterization of microorganisms and biomarker development from global ESI-MS/MS analyses of cell lysates

The capability for sensitive and accurate identification of microorganisms has potential applications that include the monitoring of industrial bioprocessing operations, food safety analyses, disease diagnosis, and detection of potential biological hazards. Efforts based upon matrix-assisted laser desorption/ionization mass spectrometry to detect and identify specific microorganisms have been actively pursued for several years. We report a new method being developed to select useful biomarkers for the identification of microorganisms based upon electrospray ionization (ESI)-ion trap mass spectrometry. Crude cell lysates are processed using a recently developed dualmicrodialysis device and then directly infused into an ion trap MS. The low ESI flow rate and precursor ion accumulation capability of the ion trap MS enables high-sensitivity MS/MS analyses. Precursor ions are automatically selected and analyzed using tandem MS (MS/MS) to produce "global" MS/MS surveys and processed to yield two-dimensional MS/MS spectral displays. Such global MS/MS surveys are demonstrated for Escherichia coli lysates. The distinctive MS/MS spectral patterns can be used to identify mass spectrometric-detected species useful as biomarkers, which then provide a basis for confident microorganism identification. The results presented demonstrate the application of this method for the identification of microorganisms, as well as for detection of bacteriophage MS2 in the presence of a large excess of E. coli.     

Liquid chromatography/mass spectrometry methods for distinguishing N-oxides from hydroxylated compounds

This study describes the application of liquid chromatography/mass spectrometry (LC/MS) methods for distinguishing between aliphatic and aromatic hydroxylations and between hydroxylations and N-oxidations. Hydroxylations and N-oxidations are common biotransformation reactions of drugs. Electrospray (ESI) and atmospheric pressure chemical ionization (APCI) were used to generate ions from liquid chromatographic effluents. ESI-MS, ESI-MS/MS, APCI-MS, and APCI-MS/MS experiments were performed on several metabolites and derivatives of loratadine (a long-acting and nonsedating tricyclic antihistamine) using an ion trap mass spectrometer (LCQ) and a triple-quadrupole mass spectrometer (TSQ). The observations are as follows: (1) LC/ESI-MS produced predominantly [M + H]+ ions with minor fragmentation. (2) LC/ESI-MS/MS data, however, showed a predominant loss of water from metabolites with aliphatic hydroxylation while the loss of water was not favored when hydroxylation was phenolic. N-Oxides (aromatic and aliphatic) showed only a small amount of water loss in the MS/MS spectra. (3) Under LC/APCI-MS conditions, aliphatic hydroxylation could be readily distinguished from aromatic hydroxylation based on the extent of water loss. In addition, N-oxides produced distinct [M + H - O]+ ions. These [M + H - O]+ ions were not produced in the APCI-MS spectra of hydroxylated metabolites. (4) Similar to the ESI-MS/MS spectra, the APCI-MS/MS spectra from the (M + H)+ ions of N-oxides yielded a small amount of water loss but no [M + H - O]+ ions. These results indicate that LC/APCI-MS can be used to distinguish between hydroxylated metabolites and N-oxides.     

Structural characterization of protein tryptic peptides via liquid chromatography/mass spectrometry and collision-induced dissociation of their doubly charged molecular ions

The formation of multiply charged molecular ions via the field-assisted ion evaporation mechanism during electrospray ionization enables the use of an atmospheric pressure ionization quadrupole mass spectrometer system for characterizing biologically important peptides. The straightforward implementation of high-performance liquid chromatography (HPLC) into this new strategy to determine the molecular weight of tryptic peptides via the pneumatically assisted electrospray (ion spray) interface is presented. Examples utilizing both microbore (1.0 mm) and standard bore (4.6 mm) inside diameter columns are shown for the LC/MS molecular weight determination of tryptic peptides in methionyl-human growth hormone (met-hGH). Injected levels from 50 to 75 pmol of tryptic digest onto 1 mm i.d. HPLC columns provided full-scan LC/MS or LC/MS/MS results without postcolumn splitting of the effluent. When standard 4.6 mm i.d. HPLC columns were used, a 20:1 postcolumn split was utilized, which required from 1 to 5 nmol of injected tryptic digest for full-scan LC/MS or LC/MS/MS results. Collision-induced dissociation (CID) mass spectra resulting from either "infusion" or on-line LC/MS/MS analysis of the abundant doubly charged ions that predominate for tryptic peptides under electrospray conditions provided structurally useful sequence information for met-hGH and human hemoglobin tryptic digests. The slower mass spectrometer scan rate used during infusion of sample provides more accurate mass assignments than on-line LC/MS or LC/MS/MS, but the latter on-line experiments preclude ambiguities caused by matrix or component interferences. However, in some instances very weak CID product ions preclude complete tryptic peptide structural characterization based upon the CID data alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Determination of ginsenosides in plant extracts from Panax ginseng and Panax quinquefolius L. by LC/MS/MS

An HPLC/MS/MS method has been developed for the characterization and quantification of ginsenosides contained in extracts of the root of Panax ginseng (Korean ginsengs) and Panax quinquefolius L. (American ginsengs). The [M + H]+ and [M + Na]+ ions were observed for ginsenoside standards (Rb1, Rb2, Rc, Rd, Re, Rf, Rg1) and four different ginseng extracts. The glycosidic linkages, the core, and the attached sugar(s) of the ginsenosides can be determined from the collision-induced dissociation spectra from the protonated molecules. The relative distribution of these ginsenosides in each extract of American or Korean ginseng was established.     

Choosing between atmospheric pressure chemical ionization and electrospray ionization interfaces for the HPLC/MS analysis of pesticides

An evaluation of over 75 pesticides by high-performance liquid chromatography/mass spectrometry (HPLC/MS) clearly shows that different classes of pesticides are more sensitive using either atmospheric pressure chemical ionization (APCI) or electrospray ionization (ESI). For example, neutral and basic pesticides (phenylureas, triazines) are more sensitive using APCI (especially positive ion). While cationic and anionic herbicides (bipyridylium ions, sulfonic acids) are more sensitive using ESI (especially negative ion). These data are expressed graphically in a figure called an ionization-continuum diagram, which shows that protonation in the gas phase (proton affinity) and polarity in solution, expressed as proton addition or subtraction (pKa), is useful in selecting APCI or ESI. Furthermore, sodium adduct formation commonly occurs using positive ion ESI but not using positive ion APCI, which reflects the different mechanisms of ionization and strengthens the usefulness of the ionization-continuum diagram. The data also show that the concept of "wrong-way around" ESI (the sensitivity of acidic pesticides in an acidic mobile phase) is a useful modification of simple PKa theory for mobile-phase selection. Finally, this finding is used to enhance the chromatographic separation of oxanilic and sulfonic acid herbicides while maintaining good sensitivity in LC/MS using ESI negative.     

Hydrophilic affinity isolation and MALDI multiple-stage tandem mass spectrometry of glycopeptides for glycoproteomics

In glycoproteomics, key structural issues, protein identification, locations of glycosylation sites, and evaluation of the glycosylation site microheterogeneity should be easily evaluated in a large number of glycoproteins, while mass spectrometry (MS) provides substantial information about individual purified glycoproteins. Considering that structural issues are elucidated by studying glycopeptides and that the tandem MS of a tryptic peptide composed of several amino acid residues is enough for protein identification, construction of an MS-based method handling tryptic glycopeptides would be of considerable benefit in research. To this end, a simple and efficient method, utilizing hydrophilic binding of carbohydrate matrixes such as cellulose and Sepharose to oligosaccharides, was successfully applied to the isolation of tryptic glycopeptides. Both peptide and oligosaccharide structures were elucidated by multiple-stage tandem MS (MS(n)) of the ions generated by matrix-assisted laser desorption/ionization (MALDI), as follows. The MALDI ion trap mass spectrum of a tryptic glycopeptide mixture from N-linked glycoproteins was composed of the [M + H]+ ions of component glycopeptides. Collision-induced dissociation (CID) of the glycopeptide [M + H]+ ion generated saccharide-spaced peaks, with an interval of, for example, 146, 162, and 203 Da, and their fragment ions corresponding to the peptide and peptide + N-acetylglucosamine (GlcNAc) species in the MS2 spectrum. The saccharide-spaced ladder served to outline oligosaccharide structures, which were then selected as precursors for subsequent MS(n) analyses. The peptide or peptide + GlcNAc ions in the MS2 spectrum or the corresponding ions abundant in the MS1 spectrum were subjected to CID for determination of peptide sequences, to identify proteins and their glycosylation sites. The strategy, isolation of glycopeptides followed by MS(n) analysis, efficiently characterized the structures of beta2-glycoprotein I with four N-glycosylation sites and was applied to an analysis of total serum glycoproteins.     

Determination of proanthocyanidins in grape products by liquid chromatography/mass spectrometric detection under low collision energy

A method has been established for the identification of proanthocyanidins and quantification of individual monoproanthocyanidins using liquid chromatography/electrospray ionization-mass spectrometric detection (LC/ESI-MSD) for raw grape products. The separated monoproanthocyanidins and oligoproanthocyanidins were individually analyzed and identified by their molecular ion peaks using LC/MS. Using HPLC/ESI-MSD, the proanthocyanidin monomers, (+)-catechin (C), (-)-epicatechin (EC), (-)-catechin gallate (CG), and (-)-epicatechin gallate (ECG) in grape products were successfully quantified by LC/MS/MS detection of protonated molecular ions and characteristic fragment ions for each component under the optimized low collision energy level of 20%. For the investigated concentration ranges of C (21.88-11,200 ng/mL), EC (21.10-10,800 ng/mL), CG (36.72-18,800 ng/mL), and ECG (39.84-20,400 ng/mL), good linearities (r2 > 0.99) for standard curves were obtained. Validation of this method showed an accuracy that was well below 15% and precision (RSD) within 8% for the four compounds. The method proposed here is simple, sensitive, and allows a direct sample preparation procedure. This is the first method that enables the determination of individual monoproanthocyanidins in grape products without any solid-phase extraction.     

ESI-MS/MS for the differentiation of diastereomeric pyrimidine glycols in mononucleosides

Pyrimidine glycols, or 5,6-dihydroxy-5,6-dihydropyrimidines, are primary lesions in DNA induced by reactive oxygen species. In this article, we report the preparation and tandem mass spectrometry (MS/MS) characterization of the two cis diastereomers of the glycol lesions of 2'-deoxyuridine, 5-methyl-2'-deoxycytidine, and thymidine. Our results show that collisional activation of the [M + Na]+ ions of all the three pairs of cis isomers and that of the [M + H]+ ions of the 2'-deoxyuridine glycols and 5-methyl-2'-deoxycytidine glycols give a facile loss of a water molecule. Interestingly, the water loss occurs more readily for the 6S isomer than for the 6R isomer. Likewise, product ion spectra of the [M - H]- ions of the two cis isomers of the 2'-deoxyuridine glycols and thymidine glycols show more facile loss of water for the 6S isomer than for the 6R isomer. MS/MS acquired at different collisional energies gave similar results, which establishes the reproducibility of spectra.     

Atmospheric pressure covalent adduct chemical ionization tandem mass spectrometry for double bond localization in monoene- and diene-containing triacylglycerols

We report a method to elucidate the structure of triacyl-glycerols (TAGs) containing monoene or diene fatty acyl groups by atmospheric pressure covalent adduct chemical ionization (APCACI) tandem mass spectrometry using acetonitrile as an adduct formation reagent. TAGs were synthesized with the structures ABB and BAB, where A is palmitate (C16:0) and B is an isomeric C18 monoene unsaturated at position 9, 11, or 13 or an isomeric diene unsaturated at positions 9 and 11, 10 and 12, or 9 and 12. In addition to the species at m/z 54 observed in previous CI studies of fatty acid methyl esters, we also found that ions at m/z 42, 81, and 95 undergo covalent reaction with TAGs containing double bonds to yield ions at m/z 40, 54, 81, and 95 units greater than that of the parent TAG: [M + 40]+, [M + 54]+, [M + 81]+, and [M + 95]+ ions. When collisionally dissociated, these ions fragment to produce two or three diagnostic ions that locate the double bonds in the TAG. In addition, ions [RCH=C=O + 40]+ and [RCH=C=O + 54]+ formed from collisional dissociation are of strong abundance in MS/MS spectra, and collisional activation of these ions produces two intense confirmatory diagnostic ions in the MS3 spectra. Fragment ions reflecting neutral loss of an sn-1-acyl group from [M + 40]+ and [M + 54]+ are more abundant than those reflecting neutral loss of an sn-2-acyl group, analogous to previous reports for protonated TAGs. The position of each acyl group on the glycerol backbone is thus determined by the relative abundances of these ions. Under the conditions in our instrument, the [M + 40]+ adduct is at the highest signal and also yields all information about the double bond position and TAG stereochemistry. With the exception of geometries about the double bonds, racemic TAG isomers containing two monoenes or dienes and a saturate can be fully characterized by APCACI-MS/MS/MS.     

Bidirectional ion transfer between quadrupole arrays: MSn ion/ion reaction experiments on a quadrupole/time-of-flight tandem mass spectrometer

Methods for bidirectional ion transmission between distinct quadrupole arrays were developed on a quadrupole/time-of-flight tandem mass spectrometer (QqTOF) containing three quadrupoles (ion guide Q0, mass filter Q1, and collision cell Q2) and a reflectron TOF analyzer, for the purpose of implementing multistage ion/ion reaction experiments. The transfer efficiency, defined as the percentage of ions detected after two transfer steps relative to the initial ion abundance, was found to be about 60% between Q2 and Q0 (with passage through the intermediate array (Q1)) and almost 100% between Q2 and Q1. Efficient ion transfer enabled new means for executing MSn experiments on an instrument of this type by operating Q1 in rf/dc mode for performing multiple steps of precursor/product ion isolation while passing ions through Q1 or trapping ions in Q1. In the latter case, the Q1 functioned as a linear ion trap. Either collision induced dissociation (CID) or ion/ion reactions can be conducted in between each stage of mass analysis. MS3 or MS4 experiments were developed to illustrate the charge increase of peptide ions via two steps of charge inversion ion/ion reactions, CID of electron-transfer dissociation (ETD) products and CID of a metal-peptide complex formed from ion/ion reactions.     

Study of the fragmentation mechanism of protonated 6-hydroxychlorzoxazone: application in simultaneous analysis of CYP2E1 activity with major human cytochrome P450s

The application of liquid chromatography tandem mass spectrometry for simultaneous analysis of major human cytochrome P450 activities via a single atmospheric pressure ionization (API) LC/MS/MS method has been hampered by the preferred detection of 6-hydroxychlorzoxazone (HCZ), the metabolite of the CYP2E1 probe, chlorzoxazone, under negative API. An initial simulation of the dissociation constants suggested the potential ionization of the enol form of HCZ at low pH, and the accurate mass measurements confirmed the presence of the protonated HCZ signal under (+) ESI at pH 3. However, the CID spectrum of the protonated HCZ resulted in a few intense, but uncommon, fragment ions that could be utilized for specific selected reaction monitoring (SRM) transitions. The deduced elemental compositions of these fragment ions indicated possible aromatic ring opening for the first two intense product ions at m/z 130 and 115, as well as chlorine radical loss for the third ion at m/z 151. Further precursor and product ion scan studies, along with the deuterium ion exchange in solution, revealed the involvement of three distinct pathways of fragmentation. The m/z 186-->130 transition, which was shown to be specific in human plasma and rat hepatic microsomes, was further combined with the SRM transition of reserpine (internal standard) and eight probe substrates for human cytochrome P450 isoforms. This led to the development of a full LC/MS/MS method capable of analyzing a total of nine human P450 activities within 3 min, including CYP2E1, using a single assay in the (+) ESI mode. The HCZ assay showed excellent linearity with a coefficient of determination (R2) greater than 0.98 at dynamic range of 0.05 (LOQ) to 40 microM. Preliminary data from the three-day validation of the HCZ assay indicated that the accuracy and precision for quality control samples was within +/- 15% of the spiked concentration at all levels.     

Electron-induced dissociation of singly charged organic cations as a tool for structural characterization of pharmaceutical type molecules

Collision-induced dissociation (CID) and electron-induced dissociation (EID) have been investigated for a selection of small, singly charged organic molecules of pharmaceutical interest. Comparison of these techniques has shown that EID carried out on an FTICR MS and CID performed on a linear ion trap MS produce complementary data. In a study of 33 molecule-cations, EID generated over 300 product ions compared to 190 product ions by CID with an average of only 3 product ions per precursor ion common to both tandem MS techniques. Even multiple stages of CID failed to generate many of the product ions observed following EID. The charge carrying species is also shown to have a very significant effect on the degree of fragmentation and types of product ion resulting from EID. Protonated species behave much like the ammonium adduct with suggestion of a hydrogen atom from the charge carrying species strongly affecting the fragmentation mechanism. Sodium and potassium are retained by nearly every product ion formed from [M + Na](+) or [M + K](+) and provide information to complement the EID of [M + H](+) or [M + NH(4)](+). In summary, EID is proven to be a fitting partner to CID in the structural elucidation of small singly charged ions and by studying EID of a molecule-ion holding different charge carrying species, an even greater depth of detail can be obtained for functional groups commonly used in synthetic chemistry.     

LC/MS/MS analyses of an oleander extract for cancer treatment

An HPLC/MS/MS method has been developed for the characterization and quantification of the cardiac glycosides oleandrin, odoroside, neritaloside and the aglycone oleandrigenin, all contained in a patented-hot-water extract of Nerium oleander L (Anvirzel). Qualitative analysis of such extracts was achieved using a hybrid tandem quadrupole time-of-flight (QqTOF) mass spectrometer. Collision-induced dissociation (CID) mass spectra of oleandrin, oleandrigenin, odoroside, and neritaloside were obtained with greater than 5 ppm mass accuracy and resolution routinely in excess of 8000 (fwhm). The detection limit for oleandrin of 20 pg (injected) was realized when the precursor-to-product ion transition, m/z 577 --> 373, was monitored. We have also applied the analytical method to the determination of oleandrin, oleandrigenin, neritaloside, and odoroside in human plasma following an intramuscular injection of Anvirzel.     

Ion trap versus low-energy beam-type collision-induced dissociation of protonated ubiquitin ions

The beam-type and ion trap collision-induced dissociation (CID) behaviors of protonated bovine ubiquitin ions were studied for charge states ranging from +6 to +12 on a modified triple quadrupole/linear ion trap tandem mass spectrometer. Both beam-type CID and ion trap CID were conducted in a high-pressure linear ion trap, followed by proton-transfer ion/ion reactions to reduce the charge states of product ions mostly to +1. The product ions observed under each activation condition were predominantly b- and y-type ions. Fragmentation patterns showed a much stronger dependence on parent ion charge state with ion trap CID than with beam-type CID using nitrogen as the collision gas, with preferential cleavages C-terminal to aspartic acid at relatively low charge states, nonspecific fragmentation at moderate charge states, and favored cleavages N-terminal to proline residues at high charge states. In the beam-type CID case, extensive cleavage along the protein backbone was noted, which yielded richer sequence information (77% of backbone amide bond cleavages) than did ion trap CID (52% of backbone amide bond cleavages). Collision gas identity and collision energy were also evaluated in terms of their effects on the beam-type CID spectrum. The use of helium as collision gas, as opposed to nitrogen, resulted in CID behavior that was sensitive to changes in collision energy. At low collision energies, the beam-type CID data resembled the ion trap CID data with preferential cleavages predominant, while at high collision energies, nonspecific fragmentation was observed with increased contributions from sequential fragmentation.     

Protein tyrosine-O-sulfation analysis by exhaustive product ion scanning with minimum collision offset in a NanoESI Q-TOF tandem mass spectrometer

Tyrosine-O-sulfated peptides were studied by nanoESI Q-TOF mass spectrometry and were found to exhibit an abundant loss of SO3 in positive ion mode under the usually nonfragmenting conditions of survey spectrum acquisition. A new strategy for the detection of tyrosine-O-sulfated peptides in total protein digests was designed based on exhaustive product ion scanning at the collision offset conditions typical for the recording of survey spectra (minimum collision offset). From these data, Q-TOF neutral loss scans for loss of 80/z and Q-TOF precursor ions scans were extracted. The specificity of this approach for analysis of tyrosine-O-sulfation was tested using a tryptic digest of bovine serum albumin spiked with sulfated hirudin (1:1 and 1000:1 molar ratio of BSA to sulfated hirudin, respectively) and using an in-solution digest of the recombinant extracellular domain of thyroid stimulating hormone receptor (ECD-TSHr). For both examples, the combination of in silico neutral loss scans for 80/z and subsequent in silico precursor ion scans resulted in a specific identification of sulfated peptides. In the analysis of recombinant ECD-TSHr, a doubly sulfated peptide could be identified in this way. Surprisingly, approximately 1/4 of the product ion spectra acquired from the tryptic digest of ECD-TSHr at minimum collision offset exhibited sequence-specific ions suitable for peptide identification. Complementary ion pairs were frequently observed, which either were b2/y(max-2) pairs or were induced by cleavage N-terminal to proline. MS/MS analysis at minimum collision offset followed by extraction of neutral loss and precursor ion scans is ideally suited for highly sensitive detection of analyte ions which exhibit facile gas-phase decomposition reactions.     

Enabling MALDI-FTICR-MS/MS for high-performance proteomics through combination of infrared and collisional activation

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) is a central tool for proteomic analysis, yet the singly protonated tryptic peptide ions produced by MALDI are significantly more difficult to dissociate for tandem mass spectrometry (MS/MS) than the corresponding multiply protonated ions. In order to overcome this limitation, current proteomic approaches using MALDI-MS/MS involve high-energy collision-induced dissociation (CID). Unfortunately, the use of high-energy CID complicates product ion spectra with a significant proportion of irrelevant fragments while also reducing mass accuracy and mass resolution. In order to address the lack of a high-resolution, high mass accuracy MALDI-MS/MS platform for proteomics, Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) and a recently developed MS/MS technique termed CIRCA (for combination of infrared and collisional activation) have been applied to proteomic analysis. Here, CIRCA is shown to be suitable for dissociating singly protonated tryptic peptides, providing greater sequence coverage than either CID or infrared multiphoton dissociation (IRMPD) alone. Furthermore, the CIRCA fragmentation spectra are of sufficient quality to allow protein identification based on the MS/MS spectra alone or in concert with the peptide mass fingerprint (PMF). This is accomplished without compromising mass accuracy or mass resolution. As a result, CIRCA serves to enable MALDI-FTICR-MS/MS for high-performance proteomics experiments.