Identification of unusual bacterial glycosylation by tandem mass spectrometry analyses of intact proteins

The characterization of protein glycosylation can be a complex and time-consuming procedure, especially for prokaryote O-linked glycoproteins, which often comprise unusual oligosaccharide structures with no known glycosylation motif. In this report, we describe a "top-down" approach that provides information on the extent of glycosylation, the molecular masses, and the structure of oligosaccharide residues on bacterial flagella, important structural proteins involved in the motility of pathogenic bacteria. Flagella from four bacterial pathogens, namely, Campylobacter jejuni, Helicobacter pylori, Aeromonas caviae, and Listeria monocytogenes, were analyzed by this top-down mass spectrometry approach. The approach needs minimal sample preparation and can be performed within a few minutes compared to the tedious and often time-consuming "bottom-up" approach involving proteolytic digestion and LC-MS-MS analyses of the suspected glycopeptides. Multiply protonated protein precursor ions subjected to low-energy collisional activation in a quadrupole time-of-flight instrument showed extensive and specific gas-phase deglycosylation resulting in the formation of abundant oxonium ions with very few fragment ions from peptidic bond cleavages. Structural information on individual carbohydrate residues is obtained using a second-generation product ion scan of oxonium ions formed by collisional activation of the intact protein ions in the source region. The four bacterial flagella examined differed not only by the extent of glycosylation but also by the nature of carbohydrate substituents. For example, the flagellin from the Gram-positive bacterium, L. monocytogenes showed O-linked GlcNAc residues at up to 6 sites/protein monomer. In contrast, the three Gram-negative bacterial pathogens C. jejuni, H. pylori and A. caviae displayed up to 19 Ser/Thr O-linked sites modified with residues structurally related to N-acetylpseudaminic acid (Pse5Ac7Ac) and in the case of Campylobacter include a novel N-acetylglutamine substituent on Pse5Am7Ac.     

Electrospray ionization tandem mass spectrometry for structural elucidation of protonated brevetoxins in red tide algae

Brevetoxins, the toxic components of "red tide" algae, all share one of two robust polycyclic ether backbone structures, but they are distinguished by differing side-chain substituents. Electrospray ionization mass spectrometry analyses of brevetoxins have shown that the polyether structure invariably has a very high affinity for sodium cations that results in the production of abundant (M + Na)+ ions even when sodium cations are only present as impurities. Because the ionic charge tends to remain localized on the sodium atom and because at least two bonds must be broken in order to produce polycyclic backbone fragmentation, it is extremely difficult to obtain abundant product ions (other than Na+) from (M + Na)+ brevetoxin precursor ions in low-energy collision-induced dissociation (CID) MS/MS experiments. This report establishes that acid additives (oxalic acid, trifluoroacetic acid, and particularly hydrochloric acid) in aqueous methanol solutions can promote high yields of protonated brevetoxin molecules (MH+ ions) for Btx-1, -2, and -9 brevetoxins. Most importantly, unlike their (M + Na)+ counterparts, MH+ precursor ions offer readily detectable product ions in CID MS/MS experiments, even under low-energy collisions. This direct structural characterization approach has provided decomposition information from brevetoxins that was previously inaccessible, including the identification of diagnostic product ions for "type A" brevetoxins (m/z 611) and "type B" brevetoxins (m/z 779, 473, 179) and characteristic ions for Btx-1 (m/z 221, 139), Btx-2 (m/z 153), and Btx-9 (m/z 157, 85). Precursor ion scans and constant neutral loss scans are proposed to enable screening of individual type A or type B brevetoxins present in naturally occurring mixtures.     

Measuring deuterium enrichment of glucose hydrogen atoms by gas chromatography/mass spectrometry

We developed a simple and accurate method for determining deuterium enrichment of glucose hydrogen atoms by electron impact gas chromatography mass spectrometry (GC/MS). First, we prepared 18 derivatives of glucose and screened over 200 glucose fragments to evaluate the accuracy and precision of mass isotopomer data for each fragment. We identified three glucose derivatives that gave six analytically useful ions: (1) glucose aldonitrile pentapropionate (m/z 173 derived from C4-C5 bond cleavage; m/z 259 from C3-C4 cleavage; m/z 284 from C4-C5 cleavage; and m/z 370 from C5-C6 cleavage); (2) glucose 1,2,5,6-di-isopropylidene propionate (m/z 301, no cleavage of glucose carbon atoms); and (3) glucose methyloxime pentapropionate (m/z 145 from C2-C3 cleavage). Deuterium enrichment at each carbon position of glucose was determined by least-squares regression of mass isotopomer distributions. The validity of the approach was tested using labeled glucose standards and carefully prepared mixtures of standards. Our method determines deuterium enrichment of glucose hydrogen atoms with an accuracy of 0.3 mol %, or better, without the use of any calibration curves or correction factors. The analysis requires only 20 μL of plasma, which makes the method applicable for studying gluconeogenesis using deuterated water in cell culture and animal experiments.     

Method for profiling mucin oligosaccharides from gastric biopsies of rhesus monkeys with and without Helicobacter pylori infection

A method for in vivo analysis of gastric mucin oligosaccharides was developed and applied to rhesus monkeys with and without Helicobacter pylori infection. Mucin-type O-linked oligosaccharides were directly released by reductive beta-elimination from gastric biopsies from rhesus monkeys. The released oligosaccharides were structurally characterized by matrix-assisted laser desorption/ionozation and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry. A diverse profile of neutral and acidic oligosaccharides was observed with these techniques. The most predominant core structure detected in all of the samples at relatively high abundance corresponded to core 2 (2HexNAc-1Hex, m/z = 611.227). The spectra generated from H. pylori-infected monkey samples showed fewer oligosaccharides collectively. Peaks corresponding to 1HexNAc-1Hex (m/z = 408.148) and 2HexNAc (m/z = 449.174), which most likely represent core structures, were absent in all infected monkeys studied, although present in all uninfected monkeys. Unsupervised cluster analysis demonstrated clear differences between the peaks detected in uninfected and naturally infected monkey samples. The results suggest that H. pylori infection is associated with lower relative abundance of oligosaccharides and loss of mucin-type core structures. This method can be applied to characterize the glycans associated with the mucin lining of live animals and allows for repeated analysis of the same animal over the course of infection.     

Measuring the mass of an electron by LC/TOF-MS: a study of "twin ions"

While investigating the in-source CID fragmentation of nonsteroidal antiinflammatory drugs (NSAIDs), it was noticed that the same fragment ion (nominal mass) formed in either positive or negative ion electrospray for a suite of NSAIDs. For example, ibuprofen with a molecular mass of 206, fragments to the m/z 161 ion in negative ion from its deprotonated molecule (m/z 205, [M - H]-) and fragments to the m/z 161 ion in positive ion from its protonated molecule (m/z 207, [M + H]+). This fragment ion was euphemistically called a "twin ion"because of the same nominal mass despite opposite charge. The CID fragmentation of the twin ions was confirmed also by LC/MS/MS ion trap. Accurate mass measurements in negative ion show that the loss was due to CO2 (measured loss of 43.9897 atomic mass units (u) versus calculated loss of 43.9898 u for N = 10) and in positive ion the loss is due to HCOOH (measured loss of 46.0048 u versus calculated loss of 46.0055 u, N = 10). It was realized that, in fact, the ions were not "identical mass twins of opposite charge" but separated in accurate mass by two electrons. The accurate mass measurement by liquid chromatography/time-of-flight-mass spectrometry (LC/TOF-MS) can distinguish between the two fragment ions of ibuprofen (161.13362 +/- 0.00019 and 161.13243 +/- 0.00014 for N = 20). This experiment was repeated for two other NSAIDs, and the mass of an electron was measured as the difference between the twin ions, which was 0.00062 u +/- 14.8% relative standard deviation (N = 20 analyses). Thus, the use of continuous calibration makes it possible to measure the mass of an electron within one significant figure using the NSAID solution. This result shows the importance of including electron mass in accurate mass measurements and the value of a benchmark test for LC/TOF-MS mass accuracy.     

Selective detection and identification of sugar nucleotides by CE-electrospray-MS and its application to bacterial metabolomics

A novel method employing CE-ESMS and precursor ion scanning was developed for the selective detection of nucleotide-activated sugars. By using precursor ion scanning for fragment ions specific to the different nucleotide carriers, i.e., ions at m/z 322 for cytidine monophosphate, m/z 323, 385, and 403 for uridine diphosphate, m/z 362, 424, and 442, for guanosine diphosphate, and m/z 346, 408, and 426 for adenosine diphosphate, it was possible to selectively detect sugar nucleotides involved in the biosynthesis of glycoconjugates such as glycoproteins and lipopolysaccharides. Enhancement of sensitivity was achieved using N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) as a sample stacking buffer and provided detection limits between 0.2 and 3.8 pmol.mL(-)(1). The present CE-ESMS method provided linear dynamic ranges over the concentrations 0.2-164 nM (r(2) = 0.952-0.997) for different nucleotide sugar standards. The application of this method is demonstrated for the identification of intracellular pools of sugar nucleotides in wild type and isogenic mutants from the bacterial pathogen Campylobacter jejuni. By using product ion scanning (with and without front-end collision-induced dissociation), it was possible to determine the precise nature of unexpected sugar nucleotides involved in the biosynthesis of pseudaminic acid, a sialic acid-like sugar previously observed on the flagellin of some pathogenic bacteria.     

Screening for disulfide bonds in proteins by MALDI in-source decay and LIFT-TOF/TOF-MS

An automated screening method is presented that uses MALDI in-source decay (MALDI-ISD) of disulfide bonds for identification of disulfide-linked peptides in MALDI mass spectra. Peptides released by ISD of a disulfide bond can be detected at an m/z ratio that corresponds to the singly protonated peptide with a reduced cysteine residue. Therefore, screening of peak lists for signal patterns that fulfill the equation, m/z (peak A) + m/z (peak B) - m/z (H2 + H+) = m/z (peak C), facilitated identification of putative ISD fragments of disulfide-linked peptides (peaks A and B) and their precursors (peak C). Signals (peak C) from putatively disulfide-linked peptides were subjected to LIFT-TOF/TOF-MS to confirm the existence of a disulfide bond. Using this method, we identified all 4 disulfide bonds in RNAseA and 8 two-disulfide clusters comprising 16 out of the 17 disulfide bonds in BSA. The presented screening method accelerated the identification of disulfide bonds in RNAseA and BSA, because the number of MS/MS spectra to be acquired was reduced by 1 order of magnitude. Less than 5% of the signals selected for LIFT-TOF/TOF-MS did not correspond to disulfide-linked peptides. Furthermore, the number of possible assignments for disulfide-linked peptides was reduced by 2-3 orders of magnitude, because knowledge of the mechanism of disulfide bond fragmentation by ISD permitted use of stricter rules for the interpretation of mass spectra. Therefore, interpretation of MS/ MS spectra of disulfide-linked peptides was considerably simplified in comparison to conventional approaches.     

Structural characterization of oligosaccharides using MALDI-TOF/TOF tandem mass spectrometry

Extensive cross-ring fragmentation ions, which are very informative of the linkages of the monosaccharide residues constituting these molecules, were readily observed in the MALDI-TOF/TOF/MS/MS spectra of oligosaccharides. These ions, in some cases, were more intense than the commonly observed Y and B ions. The A-type ions observed for the simple oligosaccharides allowed the distinction between alpha(1-4)- and alpha(1-6)-linked isobaric structures. The distinction was based not merely on the differences in the type of ions formed, but also on the ion intensities. For example, both alpha(1-4)- and alpha(1-6)-linked isobaric structures produce ions resulting from the loss of approximately 120 m/z units, but with different intensities, as a result of the fact that they correspond to two different ions (i.e., 0,4A- and 2,4A-ions), requiring different energies to be formed. Abundant A- and X-type ions were also observed for high-mannose N-glycans, allowing the determination of linkages. In addition, the high resolution furnished by MALDI-TOF/TOF allowed determination of certain ions that were commonly overlooked by MALDI-TOF or MALDI-magnetic sector instruments as a result of their lower resolution. Moreover, as a result of the fact that MS/MS spectra for parent ions and all fragment ions are acquired under the same experimental conditions, accurate determination of the molar ratios of isomeric glycans in a mixture analyzed simultaneously by MALDI-TOF/TOF tandem MS becomes possible.     

Scrubbing ions with molecules: kinetic studies of chemical noise reduction in mass spectrometry using ion-molecule reactions with dimethyl disulfide

The kinetics and product distributions of the reactions of dimethyl disulfide (DMDS) have been investigated with a group of chemical background ions commonly observed in atmospheric pressure ionization (API) mass spectrometry (MS) in order to assess the value of this molecule in filtering (or "scrubbing") these ions by changing their mass/charge (m/z) ratio. The measurements were taken with a novel electrospray ionization/selected ion flow tube/QqQ tandem mass spectrometer. The background ions studied include those with m/z 42 (protonated acetonitrile, ACN), 83 (protonated ACN dimer), 99 (protonated phosphoric acid), 117 (water cluster of m/z 99), 131 (methanol cluster of m/z 99), 149 (protonated phthalic anhydride, formed from the phthalates), and 327 (protonated triphenyl phosphate). In addition, reactions of DMDS have been studied with two model analytes--protonated caffeine and doubly protonated bradykinin--in order to assess the selectivity of DMDS reactivity. All the measurements were taken at 295 +/- 2 K in helium buffer gas at a pressure of 0.35 +/- 0.01 Torr. DMDS was observed to react efficiently with m/z 42 (ACNH+), 149 (from phthalates), and 99 (protonated phosphoric acid), with k/kc=0.91, 0.47, and 0.38, respectively. Only proton transfer was observed with ACNH+, followed by the secondary reaction of [DMDSH]+ with DMDS to yield [CH3S-S(CH3)-SCH3]+. Ligation of DMDS was the dominant primary channel observed for the reaction of the m/z 149 background ion; however, some proton transfer also was observed. Both of these primary product ions react further with DMDS to yield [CH3S-S(CH3)-SCH3]+, the structure of which we have determined computationally using DFT calculations. Only the sequential ligation with two DMDS molecules was observed for the reaction of the m/z 99 ion. Reactions of DMDS with m/z 117 [H3PO4 + H + H2O]+ and m/z 131 [H3PO4 + H + MeOH]+ were observed to proceed with k/kc=0.71 and 0.058, respectively. Ligand substitution of DMDS for H2O predominated ( approximately 94%) over DMDS ligation ( approximately 6%) in the reaction with m/z 117, while only DMDS ligation was observed for the reaction of m/z 131 with DMDS. In contrast, the reactions of DMDS with ions of m/z 83 (protonated dimer of ACN) and 327 (protonated triphenyl phosphate) were extremely inefficient, with k/kc=0.0042 and 0.0079, respectively. The higher reactivity of DMDS toward ACNH+ (m/z 42) compared to (ACN)2H+ (m/z 83) is attributed to the lower proton affinity of the unsolvated ACN. The reactivity of DMDS toward the two model analyte ions studied-protonated caffeine and doubly protonated bradykinin-was negligible, with k/kc=0.0073 and 0.010, for the respective reactions. These results suggest that, under appropriate reagent pressure conditions, DMDS can be an appropriate reagent for chemically filtering out many common API-MS background ions, without significantly affecting the observed intensity of analyte peaks.     

Improving proteomics mass accuracy by dynamic offline lock mass

Several methods to obtain low-ppm mass accuracy have been described. In particular, online or offline lock mass approaches can use background ions, produced by electrospray under ambient conditions, as calibrants. However, background ions such as protonated and ammoniated polydimethylcyclosiloxane ions have relatively weak and fluctuating intensity. To address this issue, we implemented dynamic offline lock mass (DOLM). Within every MS1 survey spectrum, DOLM dynamically selected the strongest n background ions for statistical treatments and m/z recalibration. We systematically optimized the mass profile abstraction method to find one single m/z value to represent an ion and the number of calibrants. To assess the influence of the intensity of the analyte ions, we used tandem mass spectroscopy (MS/MS) datasets obtained from MudPIT analyses of two protein samples with different dynamic ranges. DOLM outperformed both external mass calibration and offline lock mass that used predetermined calibrant ions, especially in the low-ppm range. The unique dynamic feature of DOLM was able to adapt to wide variations in calibrant intensities, leading to averaged mass error center at 0.03 ± 0.50 ppm for precursor ions. Such consistently tight mass accuracies meant that a precursor mass tolerance as low as 1.5 ppm could be used to search or filter post-search DOLM-recalibrated MS/MS datasets.     

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.     

On-line investigation of the generation of nonaqueous intermediate radical cations by electrochemistry/mass spectrometry

Anodic oxidation of triphenylamine (TPA) in acetonitrile was investigated by electrochemistry (EC) combined online with mass spectrometry (MS) through a particle beam (PB) interface, EC/PB/MS. Electrooxidation of TPA generates a TPA*+ radical cation (m/z 245) which dimerizes to tetraphenylbenzidine (TPB, MW 488). TPB is readily oxidized to TPB*+ (m/z 488) and TPB2+ (m/z 244) at the oxidation potential of TPA. In EC/PB/MS, direct monitoring of the oxidation of TPB to TPB*+ radical cation as a function of the electrode potential was achieved via selective ion monitoring of the ion peak at m/z 488. By using the relative intensity ratio of ions at m/z 244 (TPB2+) to 245 (TPA*+), the formation of TPB2+ as a function of the electrode potential was also monitored. EC/PB/MS showed a maximum rate of formation of TPB*+ at +1.2 Vvs Pd, while TPB2+ is generated at a maximum rate at +1.6 V vs Pd. The effect of spectral interference from the electron impact ionization of TPA, on EC/PB/MS results, is also discussed. Finally, a significant signal enhancement is observed in the presence of tetrabutylammonium perchlorate (TBAP) and is reported for the first time. Compatibility of coupling of EC with MS via PB interface for EC/MS studies in nonaqueous solvents is demonstrated. The observation of significant signal enhancement in the presence of TBAP may facilitate other applications of LC/MS.     

Investigation of the unusual behavior of metolachlor under chemical ionization in a hybrid 3D ion trap mass spectrometer

This article describes the strange behavior of the widely used herbicide metolachlor under chemical ionization conditions in a hybrid source ion trap mass spectrometer in gas chromatography/mass spectrometry (GC/MS) coupling. With the use of ammonia as the reagent gas, metolachlor provides a chlorinated ion at m/z 295/297, almost as abundant as the protonated molecule at m/z 284/286, which cannot be isolated to perform tandem mass spectrometry (MS(n)) experiments. Curiously, this ion at m/z = M + 12 is not observed for the herbicides acetochlor and alachlor, which present very similar chemical structures. The chemical structure of the m/z 295/297 ions and the explanation of the observed phenomenon based on the metastable behavior of these ions were elucidated on the basis of experiments including isotopic labeling and modifications of the operating conditions of the ion trap mass spectrometer. This work allows one to give new recommendations for an optimized use of hybrid source ion trap mass spectrometers.     

Development and evaluation of a candidate reference method for the determination of total cortisol in human serum using isotope dilution liquid chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry

Cortisol is an important diagnostic marker for the production of steroid hormones, and accurate measurements of serum cortisol are necessary for proper diagnosis of adrenal function. A candidate reference method involving isotope dilution coupled with liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) has been developed and critically evaluated. An isotopically labeled internal standard, cortisol-d(3), was added to serum, followed by equilibration and solid-phase and ethyl acetate extractions to prepare samples for liquid chromatography/mass spectrometry electrospray ionization (LC/MS-ESI) and liquid chromatography/tandem mass spectrometry electrospray ionization (LC/MS/MS-ESI) analyses. (M + H)(+) ions at m/z 363 and 366 for cortisol and its labeled internal standard were monitored for LC/MS. The transitions of (M + H)(+) --> [(M + H)(+) - 2H(2)O] at m/z 363 --> 327 and 366 --> 330 were monitored for LC/MS/MS. The accuracy of the measurement was evaluated by a comparison of results of this candidate reference method on lyophilized human serum reference materials for cortisol [Certified Reference Materials 192 and 193] with the certified values determined by gas chromatography/mass spectrometry reference methods and by a recovery study for the added cortisol. The results of this method for total cortisol agreed with the certified values within 1.1%. The recovery of the added cortisol ranged from 99.8% to 101.0%. This method was applied to the determination of cortisol in samples of frozen serum pools. Excellent precision was obtained with within-set CVs of 0.3%-1.5% and between-set CVs of 0.04%-0.4% for both LC/MS and LC/MS/MS analyses. The correlation coefficients of all linear regression lines ranged from 0.998 to 1.000. The detection limits (at a signal-to-noise ratio of approximately 3-5) were 10 and 15 pg for LC/MS and LC/MS/MS, respectively. This method, which demonstrates good accuracy and precision, and is free from interferences from structural analogues, qualifies as a candidate reference method and can be used as an alternative reference method to provide an accuracy base to which the routine methods can be compared.     

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.     

MALDI-TOF mass spectrometric detection of multiplex single base extended primers. A study of 17 y-chromosome single-nucleotide polymorphisms

One of the most promising techniques for typing of multiple single-nucleotide polymorphism (SNP) is detection of single base extension primers (SBE) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). We present a new MALDI-TOF MS protocol for typing of multiple SNPs in a single reaction. Biotin-labeled ddNTPs were used in the SBE reaction and solid phase-bound monomeric avidin was used as capturing/purification scheme allowing the exclusive release of the SBE products under gentle conditions using 5% triethylamine. We dubbed this method monomeric avidin triethylamine purification. The biotin-labeled ddNTPs contained linkers with different masses ensuring a clear separation of the alleles even for SBE primers with a mass of 10 300 Da. Furthermore, only 25-350 fmol of SBE primers were necessary in order to obtain reproducible MALDI-TOF spectra. Similar signal intensities were obtained in the 5500-10 300 m/z mass range by increasing the concentration of the longer SBE primers in the reaction. To validate the technique, 17 Y-chromosome SNPs were analyzed in 200 males. The precision and accuracy of the mass determination were analyzed by parametric statistic, and the potential use of MALDI-TOF MS for SNP typing is discussed.     

Multiplexed MS/MS in a quadrupole ion trap mass spectrometer

A multiplexing method for performing MS/MS on multiple peptide ions simultaneously in a quadrupole ion trap mass spectrometer (QITMS) has been developed. This method takes advantage of the inherent mass bias associated with ion accumulation in the QITMS to encode the intensity of precursor ions in a way that allows the corresponding product ions to be identified. The intensity encoding scheme utilizes the Gaussian distributions that characterize the relationship between ion intensities and rf trapping voltages during ion accumulation. This straightforward approach uses only two arbitrary waveforms, one for isolation and one for dissociation, to gather product ion spectra from N precursor ions in as little as two product ion spectra. In the example used to illustrate this method, 66% of the product ions from five different precursor peptide ions were correctly correlated using the multiplexing approach. Of the remaining 34% of the product ions, only 6% were misidentified, while 28% of the product ions failed to be identified because either they had too low intensity or they had the same m/z ratio as one of the precursor ions or the same m/z ratio as a product ion from a different precursor ion. This method has the potential to increase sample throughput, reduce total analysis times, and increase signal-to-noise ratios as compared to conventional MS/MS methods.     

Fluorescein as a versatile tag for enhanced selectivity in analyzing cysteine-containing proteins/peptides using mass spectrometry

Fluorescence-based tagging in proteomics is useful in tracking and quantifying target proteins during sample preparation or chromatographic processes. In this study, we report a novel cysteinyl tagging method using a popular fluorophore, fluorescein derivative. Such visible dyes were shown to have multiple unique characteristics, including a unique reporter ion containing the dye moiety caused by collision-induced dissociation (CID) and high affinity toward multicarboxylate functional groups, which could be useful for enhanced selectivity in MS-based proteomics. We used sulfhydryl-reactive 5-iodoacetamidofluorescein to target cysteinyl residues on the intact protein of ovalbumin and bovine serum albumin as well as proteins in MCF-7 cells. After trypsin digestion, the digests were analyzed by nanoLC-ESI-Q-TOF or MALDI-TOF. The resulting MS spectra of tryptic fragments were similar to those of unlabeled or iodoacetamide-derivatized proteins, and the MS/MS fragmentation of all fluorescein-tagged peptides was readily interpretable with intact label. Thus, fluorescein-derivatized proteins can be identified by automatic mass mapping or peptide sequencing with high confidence. It is notable that, in MS/MS mode, a strong reporter ion (m/z 422) containing the fluorescein moiety was readily detected and was believed to derive from the immonium fragment of fluorescein-labeled cysteine residues, f C (m/z 463), under CID conditions. Using a precursor scan of the reporter ion, a cysteinyl protein, ovomucoid, was identified to be present in the ovalbumin sample as an impurity. The fluorescein derivatives were further shown to have high affinities toward metal-chelating materials that have iminodiacetic acid functional groups either with or without the presence of bound metal ions. When coupling with stable isotope dimethyl labeling, fluorescein-tagged peptides could be selectively enriched, identified, and quantified. In view of its popularity, visible tracking, and unique characteristics for developing selective methods, fluorescein tagging holds great promises for targeting proteomics.     

Image charge detection mass spectrometry: pushing the envelope with sensitivity and accuracy

A novel image charge detection mass spectrometer (CDMS) with improved sensitivity and mass accuracy is described. The improved detector design and method of data analysis allow us to measure a reliable mass for a single macroion that is an order of magnitude smaller than previously achieved with CDMS. The apparatus employs an image charge detector array consisting of 22 detectors. The detectors are divided into two groups that can be floated at different potentials. The signals from the detector array are analyzed using a correlation approach to yield the velocities in the two groups of detectors and the charge. These quantities, together with the voltage difference between the two groups of detectors, provide a value for the mass. The mass, m/z, and charge distributions recorded for 300 kDa poly(ethylene oxide) (PEG) are presented. The mass distribution shows a peak at around 300 kDa with a width close to that expected from the polymer size distribution. In addition, there are broad peaks in the mass distribution at around 100 and 500 MDa. The 300 kDa ions have m/z ratios of ～2 kDa/e, and the 100 and 500 MDa ions have m/z ratios of ～40 kDa/e. The 100 and 500 MDa ions probably result from PEG aggregates that are either present in solution or the residue of large electrospray droplets.