Electron-capture dissociation tandem mass spectrometry

Electron capture dissociation (ECD) is a new fragmentation technique used in Fourier transform ion cyclotron resonance mass spectrometry and is complementary to traditional tandem mass spectrometry techniques. Disulfide bonds, normally stable to vibrational excitation, are preferentially cleaved in ECD. Fragmentation is fast and specific and labile post-translational modifications and non-covalent bonds often remain intact after backbone bond dissociation. ECD provides more extensive sequence coverage in polypeptides, and at higher electron energies even isoleucine and leucine are distinguishable. In biotechnology, the main area of ECD application is expected to be the top-down verification of DNA-predicted protein sequences, de novo sequencing, disulfide bond analysis and the combined top-down/bottom-up analysis of post-translational modifications.     

Improved collision-induced dissociation analysis of peptides by matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry through 3-sulfobenzoic acid succinimidyl ester labeling

The sulfonation reagent, a succinimidyl ester of 3-sulfobenzoic acid, has been synthesized for effective peptide sequencing. It is capable of incorporating an additional mobile proton into the peptide backbone, thus, facilitating efficient collision-induced dissociation. This reagent is easily and inexpensively prepared in short time. Tandem mass spectra of the guanidinated and reagent-sulfonated peptides consist mainly of the y-ion series with higher intensities than those observed for solely guanidinated peptides. These enhanced tandem MS attributes significantly improved MASCOT total-ion scores, thus, allowing more confident peptide sequencing. This derivatization was also very effective for the analysis of tryptic digest of human blood serum proteins separated by two-dimensional gel electrophoresis. When used in LC-MALDI/MS/MS format, this type of derivatization does not adversely affect chromatographic efficiencies.     

Defining the disulfide bonds of insulin-like growth factor-binding protein-5 by tandem mass spectrometry with electron transfer dissociation and collision-induced dissociation

The six high-affinity insulin-like growth factor-binding proteins (IGFBPs) comprise a conserved family of secreted molecules that modulate IGF actions by regulating their half-life and access to signaling receptors, and also exert biological effects that are independent of IGF binding. IGFBPs are composed of cysteine-rich amino- (N-) and carboxyl- (C-) terminal domains, along with a cysteine-poor central linker segment. IGFBP-5 is the most conserved IGFBP, and contains 18 cysteines, but only 2 of 9 putative disulfide bonds have been mapped to date. Using a mass spectrometry (MS)-based strategy combining sequential electron transfer dissociation (ETD) and collision-induced dissociation (CID) steps, in which ETD fragmentation preferentially induces cleavage of disulfide bonds, and CID provides exact disulfide linkage assignments between liberated peptides, we now have definitively mapped 5 disulfide bonds in IGFBP-5. In addition, in conjunction with ab initio molecular modeling we are able to assign the other 4 disulfide linkages to within a GCGCCXXC motif that is conserved in five IGFBPs. Because of the nature of ETD fragmentation MS experiments were performed without chemical reduction of IGFBP-5. Our results not only establish a disulfide bond map of IGFBP-5 but also define a general approach that takes advantage of the specificity of ETD and the scalability of tandem MS, and the predictive power of ab initio molecular modeling to characterize unknown disulfide linkages in proteins.     

Identification of monohydroxy progesterones produced by CYP106A2 using comparative HPLC and electrospray ionisation collision-induced dissociation mass spectrometry

Two previously uncharacterised products, produced by recombinant CYP106A2 of Bacillus megaterium ATCC 13368 using progesterone as substrate, were identified. For this purpose a combination of comparative HPLC and electrospray ionisation collision induced dissociation mass spectrometry (ESI CID MS) was established and applied for rapid identification of the steroids, which were identified as 11alpha-hydroxyprogesterone and 9alpha-hydroxyprogesterone. The pharmaceutical relevance of these steroids is discussed. Furthermore, the hydroxylation activity was quantified for all monohydroxylation products (15beta-hydroxyprogesterone, 6beta-hydroxyprogesterone, 11alpha-hydroxyprogesterone, and 9alpha-hydroxyprogesterone). The V(max) values for 15beta-hydroxyprogesterone, 6beta-hydroxyprogesterone, 11alpha-hydroxyprogesterone, and 9alpha-hydroxyprogesterone were determined as 337.3+/-43.7, 22.3+/-0.9, 17.5+/-0.9, and 6.5+/-0.3nmol product/min/nmol CYP106A2, respectively.     

Shotgun collision-induced dissociation of peptides using a time of flight mass analyzer

Parallel collision-induced dissociation (CID) of peptides rather than serial, as is customary, results in loss of the obvious parent-fragment ion lineage available from CID on a single ion. We report proof-of-principle results suggesting the feasibility of parallel peptide CID, referred to here as shotgun CID, for protein identification when using the measured mass accuracies available from a time of flight mass analyzer and currently available search routines such as SEQUEST. Additionally, we report that parent-fragment ion lineage may be reconstructed from information encoded in the chromatographic single ion current traces of peptides.     

Identification of carbonylated proteins from enriched rat skeletal muscle mitochondria using affinity chromatography-stable isotope labeling and tandem mass spectrometry

We describe a strategy for the identification of carbonylated proteins from complex protein mixtures that combines biotin hydrazide labeling of protein carbonyl groups, avidin affinity chromatography, multiplexed iTRAQ reagent stable isotope labeling, and analysis using pulsed Q dissociation (PQD) operation on an LTQ linear ion trap mass spectrometer. This strategy provided the ability to distinguish biotin hydrazide labeled, avidin purified, carbonylated proteins from non-carbonylated background proteins with affinity for the avidin column, derived from a control sample. Applying this strategy to the identification of crudely enriched rat skeletal muscle mitochondrial protein isolates, we generated a catalogue of over 200 carbonylated proteins by virtue of their quantitative enrichment compared to the control sample. The catalogue contains many mitochondrial localized proteins shown to be susceptible to carbonyl modification for the first time, including numerous transmembrane proteins involved in oxidative phosphorylation. Other oxidative modifications (e.g. nitrosylation, hydroxylation) were also identified on many of the carbonylated proteins, providing further evidence of the susceptibility of these proteins to oxidative damage. The results also demonstrate the utility of PQD operation on the LTQ instrument for quantitative analysis of iTRAQ reagent-labeled peptide mixtures, as well as the quantitative reproducibility of the avidin-affinity enrichment method.     

Quantification of acetylation at proximal lysine residues using isotopic labeling and tandem mass spectrometry

With the emergence of the histone code as a key determinant in the regulation of gene expression, it has been important to develop tools that can not only identify the types and locations of myriad modifications, but also determine how the levels of these modifications change as a result of various processes in a cell. Mass spectrometry has become a method of choice for the investigation of post-translational modifications in histone proteins. Described in this article is a mass spectrometric method that is useful for direct quantification of levels of acetylation at lysines residues in close proximity to one another, as is the case for the amino terminal tail of histone H4. This method involves fragmentation of peptides into b and y ions that contain one or more sites of modification and isotopic labeling which ensures equivalent ionization and fragmentation.     

Detection of phosphodiester adducts formed by the reaction of benzo[a]pyrene diol epoxide with 2'-deoxynucleotides using collision-induced dissociation electrospray ionization tandem mass spectrometry

In this study, we investigated the products formed following the reaction of benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (B[a]PDE) with 2′-deoxynucleoside 3′-monophosphates. The B[a]PDE plus 2′-deoxynucleotide reaction mixtures were purified using solid phase extraction (SPE) and subjected to HPLC with fluorescence detection. Fractions corresponding to reaction product peaks were collected and desalted using SPE prior to analysis for the presence of molecular ions corresponding to m/z 648, 632, 608 and 623 [M−H]⁻ consistent with B[a]PDE adducted (either on the base or phosphate group) 2′-deoxynucleotides of guanine, adenine, cytosine and thymine, respectively, using LC-ESI-MS/MS collision-induced dissociation (CID). Reaction products were identified having CID product ion spectra containing product ions at m/z 452, 436 and 412 [(B[a]Ptriol+base)−H]⁻, resulting from cleavage of the glycosidic bond between the 2′-deoxyribose and base, corresponding to B[a]PDE adducts of guanine, adenine and cytosine, respectively. Further reaction products were identified having unique CID product ion spectra characteristic of B[a]PDE adduct formation with the phosphate group of the 2′-deoxynucleotide. The presence of product ions at m/z 399 and 497 were observed for all four 2′-deoxynucleotides, corresponding to [(B[a]Ptriol+phosphate)−H]⁻ and [(2′-deoxyribose+phosphate+B[a]Ptriol)−H]⁻, respectively. In conclusion, this investigation provides the first direct evidence for the formation of phosphodiester adducts by B[a]PDE following reaction with 2′-deoxynucleotides.     

Determination of sitagliptinin human plasma using protein precipitation and tandem mass spectrometry

A simple offline LC–MS/MS method for the quantification of sitagliptin in human plasma is described. Samples are prepared using protein precipitation. Filtration of the supernatants through a Hybrid-SPE-PPT plate was found to be necessary to reduce ionization suppression caused by co-elution of phospholipids with sitagliptin. The sitagliptin and its stable isotope labeled internal standard (IS) were chromatographed under hydrophilic interaction chromatography conditions on a Waters Atlantis HILIC Silica column (2.1 mm × 50 mm, 3 μm) using a mobile phase of ACN/H₂O (80/20, v/v) containing 10 mM NH₄Ac (pH 4.7). The sample drying after protein precipitation due to high organic content in the sample is not necessary, because HILIC column was used. The analytes were detected with a tandem mass spectrometer employing a turbo ion spray (TIS) interface in positive ionization mode. The multiple reaction monitoring (MRM) transitions were m/z 408 → 235 for sitagliptin and m/z 412 → 239 for IS. The lower limit of quantitation (LLOQ) for this method is 1 ng/mL when 100 μL of plasma is processed. The linear calibration range is 1–1000 ng/mL for sitagliptin. Intra-day precision and accuracy were assessed based on the analysis of six sets of calibration standards prepared in six lots of human control plasma. Intra-day precision (RSD%, n = 6) ranged from 1.2% to 6.1% and the intra-day accuracy ranged from 97.6% to 103% of nominal values.     

Identification of oxidized proteins in rat plasma using avidin chromatography and tandem mass spectrometry

The objective in much of the proteomics literature today is to establish the difference between healthy and disease states at the protein level using blood plasma. A critical component in this endeavor is to establish what is normal. The focus of the work reported here was to do this with oxidized proteins that might relate to oxidative stress and oxidative stress-related diseases. Oxidative stress is known to increase markedly in cancer, diabetes, heart disease, and neurodegenerative diseases. Since proteins are one of the targets of ROS, generated by oxidative stress, oxidized proteins are excellent biomarker candidates for these diseases. But first it is necessary to identify oxidized proteins that occur in the healthy state. Healthy rat plasma was used in this study as a source for the identification of naturally oxidized proteins. Freshly drawn blood was treated with biotin hydrazide to selectively derivatize carbonyl groups in oxidized proteins. Oxidized proteins thus biotinylated were separated from the other plasma proteins using avidin affinity chromatography. Affinity selected proteins were further fractionated on a C(8) RP column and fractions collected. The collected fractions were then tryptic digested and the peptides identified using a combination of LC/MS/MS and database searches. One hundred forty-six proteins were identified using 700 signature peptides from the tryptic digested chromatographic fractions. The most frequently encountered proteins in the samples were keratins. Brain and liver were among the organs contributing the most oxidized proteins to plasma followed by heart and kidney.     

EBP, a program for protein identification using multiple tandem mass spectrometry datasets

MS/MS combined with database search methods can identify the proteins present in complex mixtures. High throughput methods that infer probable peptide sequences from enzymatically digested protein samples create a challenge in how best to aggregate the evidence for candidate proteins. Typically the results of multiple technical and/or biological replicate experiments must be combined to maximize sensitivity. We present a statistical method for estimating probabilities of protein expression that integrates peptide sequence identifications from multiple search algorithms and replicate experimental runs. The method was applied to create a repository of 797 non-homologous zebrafish (Danio rerio) proteins, at an empirically validated false identification rate under 1%, as a resource for the development of targeted quantitative proteomics assays. We have implemented this statistical method as an analytic module that can be integrated with an existing suite of open-source proteomics software.     

Identification of multiple folding pathways of monellin using pulsed thiol labeling and mass spectrometry

Protein folding reactions often display multiexponential kinetics of changes in intrinsic optical signals, as a manifestation of heterogeneity, either on one folding pathway or on multiple folding pathways. Delineating the origin of this heterogeneity is difficult because different coexisting structural forms of a protein cannot be easily distinguished by optical probes. In this study, the complex folding reaction of single-chain monellin has been investigated using a pulsed thiol labeling (SX) methodology in conjunction with mass spectrometry, which measures the kinetics of burial of a cysteine side chain thiol during folding. Because it can directly distinguish between unfolded and folded molecules and can measure the disappearance of the former during folding, the pulsed SX methodology is an ideal method for investigating whether multiple pathways are operative during folding. The kinetics of burial of the C42 thiol of monellin was observed to follow biexponential kinetics. To determine whether this was because the fast phase leads to the partial protection of the thiol group in all the molecules or to complete protection in only a fraction of the molecules, the duration and intensity of the labeling pulse were varied. The observation that the extent of labeling did not vary with the duration of the pulse cannot be explained by a simple sequential folding mechanism. Two parallel folding pathways are shown to be operative, with one leading to the formation of thiol-protective structure more rapidly than the other.     

Matrix-assisted laser desorption/ionization mass spectrometry of neutral and acidic oligosaccharides with collision-induced dissociation

Using ribonuclease B and human alpha 1-acid glycoprotein (AGP) as model glycoproteins, matrix-assisted laser desorption/ionization (MALDI) mass spectrometry with collision-induced dissociation (CID) is validated here as an effective tool for oligosaccharide sequencing. The spectra acquired for high-mannose and complex oligosaccharide structures show characteristic fragments resulting from cleavages of the glycosidic bonds and a few cross-ring cleavages. Esterification of the sialic acid residues is essential in stabilizing the acidic N-linked oligosaccharides. An important analytical feature observed in all acquired spectra is the occurrence of cleavages on the same antenna up to the branching point, as deduced from the absence of fragmentation due to the simultaneous cleavages on two or more antennas.     

Identification of hyperreactive cysteines within ryanodine receptor type 1 by mass spectrometry

The skeletal-type ryanodine receptor (RyR1) undergoes covalent adduction by nitric oxide (NO), redox-induced shifts in cation regulation, and non-covalent interactions driven by the transmembrane redox potential that enable redox sensing. Tight redox regulation of RyR1 is thought to be primarily mediated through highly reactive (hyperreactive) cysteines. Of the 100 cysteines per subunit of RyR1, approximately 25-50 are reduced, with 6-8 considered hyperreactive. Thus far, only Cys-3635, which undergoes selective adduction by NO, has been identified. In this report, RyR1-enriched junctional sarcoplasmic reticulum is labeled with 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM, 1 pmol/microg of protein) in the presence of 10 mm Mg(2+), conditions previously shown to selectively label hyperreactive sulfhydryls and eliminate redox sensing. The CPM-adducted RyR1 is separated by gel electrophoresis and subjected to in-gel tryptic digestion. Isolation of CPM-adducted peptides is achieved by analytical and microbore high-performance liquid chromatography utilizing fluorescence and UV detection. Subsequent analysis using two direct and one tandem mass spectrometry methods results in peptide masses and sequence data that, compared with the known primary sequence of RyR1, enable unequivocal identification of CPM-adducted cysteines. This work is the first to directly identify seven hyperreactive cysteines: 1040, 1303, 2436, 2565, 2606, 2611, and 3635 of RyR1. In addition to Cys-3635, the nitrosylation site, six additional cysteines may contribute toward redox regulation of the RyR1 complex.     

Identification of Polygonum orientale constituents using high-performance liquid chromatography high-resolution tandem mass spectrometry

Our primary focus in this research was to identify and characterize its bioactive compounds for potential therapeutic use. Twenty-seven metabolites of Polygonum orientale were identified using LC-QTOF tandem mass spectrometry. Interestingly, P. orientale extracts included several highly oxygenated flavonoids were isolated from P. orientale by column chromatography. ¹³C NMR data of highly oxygenated flavonoids (1–7) are reported here for the first time. In addition, nitric oxide, 1,1-diphenyl-2-picrylhydrazyl, and water-soluble tetrazolium salt assays were carried out on the isolated compounds to investigate their anti-inflammatory, anti-oxidant, and neuroprotective activities, respectively. Compounds 1, 2, 3, 5, 7, and 8 significantly attenuated lipopolysaccharide-stimulated NO production in BV2 cells without affecting cell viability. Compounds 9–12 exhibited significant antioxidant activity, while compounds 8, 9, and 12 exhibited protective effects against glutamate-induced neurotoxicity in HT22 cells. Our results indicate that P. orientale is a promising source of natural agents for the potential treatment of inflammation and neurodegenerative diseases.     

The identification of cyclic nucleotides from living systems using collision-induced dissociation of ions generated by fast atom bombardment mass spectrometry

Extracts derived from rat liver and Phaseolus leaves are shown, by collision-induced dissociation of [MH]+ ions generated by fast atom bombardment mass spectrometry, to contain cytidine 3',5'-cyclic monophosphate and guanosine 3',5'-cyclic monophosphate respectively, and not the 2',3'-cyclic isomers. Interference peaks, expected to be common to all mass-analysed ion kinetic energy spectra of ions generated by the fast atom bombardment process from glycerol-based matrices are identified. It is shown that unequivocal identification of cytidine 3',5'-cyclic monophosphate can be made at the microgram level. Attempts to derive a quantitative procedure based on using different cyclic nucleotides as internal standards were unsuccessful due to the poor solubility of these compounds in the matrix system.     

Laser-induced dissociation of phosphorylated peptides using matrix assisted laser desorption/ionization tandem time-of-flight mass spectrometry

Reversible phosphorylation is one of the most important posttranslational modifications of cellular proteins. Mass spectrometry is a widely used technique in the characterization of phosphorylated proteins and peptides. Similar to nonmodified peptides, sequence information for phosphopeptides digested from proteins can be obtained by tandem mass analysis using either electrospray ionization or matrix assisted laser desorption/ionization (MALDI) mass spectrometry. However, the facile loss of neutral phosphoric acid (H₃PO₄) or HPO₃ from precursor ions and fragment ions hampers the precise determination of phosphorylation site, particularly if more than one potential phosphorylation site or concensus sequence is present in a given tryptic peptide. Here, we investigated the fragmentation of phosphorylated peptides under laser-induced dissociation (LID) using a MALDI-time-of-flight mass spectrometer with a curved-field reflectron. Our data demonstrated that intact fragments bearing phosphorylated residues were produced from all tested peptides that contain at least one and up to four phosphorylation sites at serine, threonine, or tyrosine residues. In addition, the LID of phosphopeptides derivatized by N-terminal sulfonation yields simplified MS/MS spectra, suggesting the combination of these two types of spectra could provide an effective approach to the characterization of proteins modified by phosphorylation.