Isotope pattern vector based tandem mass spectral data calibration for improved peptide and protein identification

Tandem mass spectra contain noisy peaks which make peak picking for peptide identification difficult. Moreover, all spectral peaks can be shifted due to systematic measurement errors. In this paper, a novel use of an isotope pattern vector (IPV) is proposed for denoising and systematic measurement error prediction. By matching the experimental IPVs with the theoretical IPVs of candidate fragment ions, true ionic peaks can be identified. Furthermore, these identified experimental IPVs and their corresponding theoretical IPVs are used in an optimization process to predict the systematic measurement error associated with the target spectrum. In return, the subsequent spectral data calibration based on the predicted systematic measurement error enhances the data quality. We show that such an integrated denoising and calibration process leads to significantly improved peptide and protein identification. Different from the commonly employed chemical calibration methods, our IPV‐based method is a purely computational method for individual spectra analysis and globally optimizes the use of spectral data. Copyright © 2009 John Wiley & Sons, Ltd.     

Chimera Spectrum Diagnostics for Peptides Using Two-Dimensional Partial Covariance Mass Spectrometry

The rate of successful identification of peptide sequences by tandem mass spectrometry (MS/MS) is adversely affected by the common occurrence of co-isolation and co-fragmentation of two or more isobaric or isomeric parent ions. This results in so-called `chimera spectra’, which feature peaks of the fragment ions from more than a single precursor ion. The totality of the fragment ion peaks in chimera spectra cannot be assigned to a single peptide sequence, which contradicts a fundamental assumption of the standard automated MS/MS spectra analysis tools, such as protein database search engines. This calls for a diagnostic method able to identify chimera spectra to single out the cases where this assumption is not valid. Here, we demonstrate that, within the recently developed two-dimensional partial covariance mass spectrometry (2D-PC-MS), it is possible to reliably identify chimera spectra directly from the two-dimensional fragment ion spectrum, irrespective of whether the co-isolated peptide ions are isobaric up to a finite mass accuracy or isomeric. We introduce ‘3-57 chimera tag’ technique for chimera spectrum diagnostics based on 2D-PC-MS and perform numerical simulations to examine its efficiency. We experimentally demonstrate the detection of a mixture of two isomeric parent ions, even under conditions when one isomeric peptide is at one five-hundredth of the molar concentration of the second isomer.     

Advantages of using nested collision induced dissociation/post-source decay with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry: sequencing of novel peptides from wasp venom

We have examined the applicability of the 'nested' collision induced dissociation/post-source decay (CID/PSD) method to the sequencing of novel peptides from solitary wasps which have neurotoxic venom for paralyzing other insects. The CID/PSD spectrum of a ladder peptide derived from an exopeptidase digest was compared with that of the intact peptide. The mass peaks observed only in the CID/PSD spectrum of a ladder peptide were extracted as C-terminal fragment ions. Assignment of C-terminal fragment ions enabled calculation of N-terminal fragment masses, leading to differentiation between N-terminal fragment ions and internal fragment ions. This methodology allowed rapid and sensitive identification by removing ambiguity in the assignment of the fragment ions, and proved useful for sequencing unknown peptides, in particular those available as natural products with a limited supply.     

De novo sequencing of tryptic peptides sulfonated by 4-sulfophenyl isothiocyanate for unambiguous protein identification using post-source decay matrix-assisted laser desorption/ionization mass spectrometry

A simple method of solid-phase derivatization and sequencing of tryptic peptides has been developed for rapid and unambiguous identification of spots on two-dimensional gels using post-source decay (PSD) matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The proteolytic digests of proteins are chemically modified by 4-sulfophenyl isothiocyanate. The derivatization reaction introduces a negative sulfonic acid group at the N-terminus of a peptide, which can increase the efficiency of PSD fragmentation and enable the selective detection of only a single series of fragment ions (y-ions). This chemically assisted method avoids the limitation of high background normally observed in MALDI-PSD spectra, and makes the spectra easier to interpret and facilitates de novo sequencing of internal fragment. The modification reaction is conducted in C(18) microZipTips to decrease the background and to enhance the signal/noise. Derivatization procedures were optimized for MALDI-PSD to increase the structural information and to obtain a complete peptide sequence even in critical cases. The MALDI-PSD mass spectra of two model peptides and their sulfonated derivatives are compared. For some proteins unambiguous identification could be achieved by MALDI-PSD sequencing of derivatized peptides obtained from in-gel digests of phosphorylase B and proteins of hepatic satellite cells (HSC).     

Backbone cleavages of [M - H](-) anions of peptides. Cyclisation of citropin 1 peptides involving reactions between the C-terminal [CONH](-) residue and backbone amide carbonyl groups. A new type of beta cleavage: a joint experimental and theoretical study

This paper reports the study of backbone cleavages in the collision-induced negative-ion mass spectra of the [M - H](-) anions of some synthetic modifications of the bioactive amphibian peptide citropin 1 (GLFDVIKKVASVIGGL-NH(2)). The peptides chosen for study contain no amino acid residues which could effect facile side-chain cleavage, i.e. Ser (-CH(2)O, side-chain cleavage) and Asp (-H(2)O) are replaced by Ala or Lys. We expected that such peptides should exhibit standard and pronounced peaks due to alpha cleavage ions (and to a lesser extent beta cleavage ions) in their collision-induced negative-ion spectra. This expectation was realised, but the spectra also contained peaks formed by a new series of cleavage anions. These are produced following cyclisation of the C-terminal CONH(-) moiety at carbonyl functions of amide groups along the peptide backbone; effectively transferring the NH of the C-terminal CONH(-) group to other amino acid residues. We have called the product anions of these processes beta' ions, in order to distinguish them from standard beta ions. Some beta' ions also fragment directly to some other beta' ions of smaller mass. The reaction coordinates of alpha,beta and beta' backbone processes have been calculated at the HF/6-31G*//AM1 level theory for simple model systems. The initial cyclisation step of the beta' sequence is barrierless and exothermic. Subsequent steps have a maximum barrier of +40 kcal mol(-1), with the overall reaction being endothermic by some 30 kcal mol(-1) at the level of theory used. These calculations take no account of the complexity of the conformationally flexible peptide system, and it is surprising that each of the two reacting centres can 'find' each other in such a large system.     

High sequence-coverage detection of proteolytic peptides using a bis(terpyridine)ruthenium(II) complex

The use of a bis(terpyridine)ruthenium(ii) complex for peptide labeling (Ru-CO labeling) supplied high intensity peaks in mass spectrometry (MS) analysis that overcame the contribution of protonation or sodiated adduction to peptides. Ru-CO-labeled insulin A- and B-chains were detected simultaneously in comparable peak abundance by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The mass spectra of chymotryptic peptide fragments of Ru-CO-labeled insulin also simultaneously indicated both N-terminal fragment ions, and amino acid sequences were determined easily by matrix-assisted laser desorption/ionization post-source-decay (MALDI-PSD). The sensitivity of detecting Ru-CO-labeled peptide fragment ions was not dependent on the length or the sequences of the peptides. The Ru-CO labeling method was applied to tryptic myoglobin fragments. The method indicated that each fragment ion is detected nearly equal in abundance and enabled the desired fragment ions to be distinguished from matrix clusters or their in-source fragments in lower mass regions. The desired fragment ions can be found in the mass region higher than 670.70 (= Ru-CO). This method provided a high sequence coverage (96%) by peptide mass fingerprinting (PMF). Application of this method to a protein mixture (myoglobin, lysozyme and ubiquitin) successfully achieved high sequence-coverage characterization (>90%) of these proteins simultaneously.     

Mass spectrometry of 4-amino-4′-nitroazobenzene compounds

The mass spectra of 32 substituted 4-amino-4′-nitroazobenzene compounds have been recorded and the most intense peaks have been used to characterize these spectra. It was found that the spectra of 4-amino-4′-nitroazobenzene compounds are characterized by peaks due to: (1) molecular ions, (2) fragment ions formed by cleavage of one of the carbon-nitrogen bonds adjacent to the azo linkage with the positive charge remaining with the amine fragment, (3) ions formed by cleavage alpha to the amine nitrogen with the charge remaining with the amine substituent, (4) ions formed by cleavage beta to the amine nitrogen with the loss of the amine substituent fragment, (5) secondary ions formed by cleavage beta to the amine nitrogen with the loss of the amine substituent fragment from the primary amine fragment (2), and (6) ions formed by loss of NO from the molecular ion. This work shows that 4-amino-4′-nitroazobenzene compounds exhibit fragmentation which is dependent in a consistent manner on the types of substituents. This work provides a basis for a systematic approach to the identification of 4-amino-4′-nitroazobenzene compounds.     

Development of novel guanidino-labelling derivatisation (GLaD) reagents for liquid chromatography/matrix-assisted laser desorption/ionisation analysis

A new generation of guanidino-labelling (GLaD) reagents were developed for quantitative proteomics using offline microcapillary liquid chromatography (LC) matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS). In order to reduce the unwanted overlapping between the isotopic envelopes of the two differentially labelled peptide ions, a novel synthetic route was described for production of both (13)C- and (15)N-containing isotopomers of N,O-dimethylisourea. The use of these types of isotopes has no deleterious effect on the retention times of both differentially labelled peptides during offline microbore reversed-phase LC. In addition, the possibility to incorporate a mass difference of 4 Da can be exploited during post-source decay analysis to generate product ion spectra in which fragment ions containing the modifications appear as doublets in the corresponding product ion spectra, thus facilitating identification of the C-terminal fragment ions.     

Protein desolvation in UV matrix-assisted laser desorption/ionization (MALDI)

We describe experiments in MALDI-TOF and MALDI-TOF-TOF showing that the ejection of protein-matrix cluster ions and their partial decay in the source occur in MALDI. The use of radial beam deflection and small size detector in linear mode allows detection of ions with higher time-of-flight and kinetic energy deficit. MALDI-TOF-TOF experiments were carried out by selecting chemical noise ions at m/z higher than that of a free peptide ion. Whatever the selected m/z (up to m/z 300) the molecular peptide ion appeared as the main fragment. The production of protein-matrix clusters and their partial decay in the source was found to increase with the size of the protein (MW from 1000 to 150,000 u), although it decreases with increasing charge state. These effects were observed for different matrices (HCCA and SA) and in a large laser fluence range. Experimental results and calculation highlight that a continuous decay of protein-matrix cluster ions occurs in the source. This decay-desolvation process can account for the high-mass tailing and peak shifting as well as the strong noise/background in the mass spectra of proteins.     

Charge state separation for protein applications using a quadrupole time-of-flight mass spectrometer

A novel method for separating ions according to their charge state using a quadrupole time-of-flight mass spectrometer is presented. The benefits of charge state separation are particularly apparent in protein identification applications at low femtomole concentration levels, where in conventional TOF MS spectra peptide ions are often lost in a sea of chemical noise. When doubly and triply charged tryptic peptide ions need to be filtered from singly charged background ions, the latter are suppressed by two to three orders of magnitude, while from 10-50% of multiply charged ions remain. The suppression of chemical noise reduces the need for chromatography and can make this experimental approach the electrospray equivalent of conventional MALDI peptide maps. If unambiguous identification cannot be achieved, MS/MS experiments are performed on the precursor ions identified through charge separation, while the previously described Q2-trapping duty cycle enhancement is tuned for approximately 1.4 of the precursor m/z to enhance intensities of ions with m/z values above that of the precursor. The resulting product ion spectra contain few fragments of impurities and provide quick and unambiguous identification through database search. The multiple charge separation technique requires minimal tuning and may become a useful tool for analysis of complex mixtures.     

Quantitative TOF-SIMS analysis of oligomeric degradation products at the surface of biodegradable poly(alpha-hydroxy acid)s

This paper reports the development of a new method for quantification of the hydrolytic surface degradation kinetics of biodegradable poly(alpha-hydroxy acid)s using time-of-flight secondary ion mass spectrometry (TOF-SIMS). We report results from static SIMS spectra of a series of poly(alpha-hydroxy acid)s including poly(glycolic acid), poly(L-lactic acid), and random poly(D,L-lactic acid-co-glycolic acid) hydrolyzed in various buffer systems. The distribution of the most intense peak intensities of ions generated in high mass range of the spectrum reflects the intact degradation products (oligomeric hydrolysis products) of each biodegradable polymer. First, a detailed analysis of the oligomeric ions is given based on rearrangement of the intact hydrolysis products. The pattern of ions can distinguish both degradation-generated intact oligomers and their fragment ion peaks with a variety of combinations of each repeat unit. Then, the integration and summation of the area of all ion peaks with the same number of repeat units is proposed as a measurement that provides a more accurate MW average than the typically used method which counts only the most intense peak. The multiple ion summation method described in this paper would be practical in the improvement of quantitative TOF-SIMS studies as a better data reduction method, especially in the surface degradation kinetics of biodegradable polymers.     

Improved peptide sequencing using isotope information inherent in tandem mass spectra

We demonstrate here the use of natural isotopic 'labels' in peptides to aid in the identification of peptides with a de novo algorithm. Using data from ion trap tandem mass spectrometric (MS/MS) analysis of 102 tryptic peptides, we have analyzed multiple series of peaks within LCQ MS/MS spectra that 'spell' peptide sequences. Isotopic peaks from naturally abundant isotopes are particularly prominent even after peak centroiding on y- and b-series ions and lead to increased confidence in the identification of the precursor peptides. Sequence analysis of the MS/MS data is accomplished by finding sequences and subsequences in a hierarchical manner within the spectra.     

Noise filtering techniques for electrospray quadrupole time of flight mass spectra

The sensitivity of protein identification by peptide sequencing using a nanoelectrospray ion source is limited by our ability to identify peptide ions in the mass spectrum. Their intensity must be higher than the chemical noise level to allow a rapid localization in the spectrum. Multiply-charged peptide ions on or below this level can only be found because their isotopic pattern is denser than that of the mostly singly-charged chemical background ions. However, to find peptides by looking for multiply-charged isotope clusters can be very timeconsuming and may lead to misassignments of the first isotope. Here we present a software-based method to increase the signal to noise ratio of ion signals in an electrospray spectrum. The software has two elements, one to reduce the noise level and a second to increase the intensity of ion peaks. Both methods together generate a spectrum in which the signal to noise ratio of ion signals is considerably improved. Peptide ions previously hidden in the chemical background are dismantled and can now be localized and selected for fragmentation. The method has been used successfully to identify low level proteins separated by one dimensional gel electrophoresis.     

Computerized separation of chromatographically unresolved peaks

A computerized peak deconvolution software and mass spectra were successfully applied for the deconvolution of overlapped peak cluster in the chromatogram obtained separating the complex mixture of pesticides by retention time locking gas chromatography-mass spectroscopy. The method based on the unique fragment ions in the spectra can be used for deconvolution of peak clusters if mass spectra of overlapped peaks differ. This method allows determining actual retention times of overlapped peaks. Peak areas found by this method however, cannot be used naturally for the quantitative purposes as the abundance of fragment ions used for this deconvolution procedure can dramatically differ. Computer assisted deconvolution of peaks in the peak clusters gives more realistic peak area ratios as at this method it is supposed equal response for all peaks overlapped in a cluster.     

A comparison of electron ionization mass spectra obtained at 70 eV,low electron energies,and with cold EI and their NIST library identification probabilities

Electron ionization (EI) mass spectra of 46 compounds from several different compound classes were measured. Their molecular ion abundances were compared as obtained with 70‐eV EI, with low eV EI (such as 14 eV), and with EI mass spectra of vibrationally cold molecules in supersonic molecular beams (Cold EI). We further compared these mass spectra in their National Institute of Standards and Technology (NIST) library identification probabilities. We found that

1. Low eV EI is not a soft ionization method, and it has little or no influence on the molecular ion relative abundances for large molecules and those with weak or no molecular ions.
2. Low eV EI for compounds with abundant or dominant molecular ions in their 70 eV mass spectra results in the reduction of low mass fragment ions abundances thereby reducing their NIST library identification probabilities thus rarely justifies its use in real‐world applications.
3. Cold EI significantly enhances the relative abundance of the molecular ions particularly for large compounds; yet, it retains the low mass fragment ions; hence, Cold EI mass spectra can be effectively identified by the NIST library.
4. Different standard EI ion sources provide different 70 eV EI mass spectra. Among the Agilent technologies ion sources, the “Extractor” exhibits relatively abundant molecular ions compared with the “Inert” ion source, while the “High efficiency source” (HES) provides mass spectra with depleted molecular ions compared with the “Inert” ion source or NIST library mass spectra.

These conclusions are demonstrated and supported by experimental data in nine figures and two tables.     

Formation of M+. ions under matrix fast atom bombardment conditions: Does charge exchange reaction occur?

The formation of molecular ions, M^+., under fast atom bombardment (FAB) conditions using a liquid matrix was examined by using a new type of synthesized compounds in which preferential M^+. peaks appear in their FAB spectra. The FAB spectra were compared with the corresponding mass spectra obtained by the electron impact (EI) ionization, chemical ionization (CI) and charge-exchange ionization (CEI) methods. All of the spectra showed preferential peaks of M^+. ion and a characteristic intense fragment ion peak originating from a β-fission. The FAB spectra were similar in the fragment ions appearing in the EI spectra and were very similar in the fragmentation pattern to the CEI spectra using Ar^+. and Xe^+. as the reagent ions. Further, the FAB spectra did not show any doubly charged ion peaks, while the 70 eV EI spectra showed the peaks of doubly charged molecular and/or fragment ions. The isobutane CI spectra of the synthesized compounds suggested that the formation of M^+. ions occurred through the CE reaction with isobutane ion, C₄H₁₀^+., and the CI spectra showed a marked intense fragment ion peak originating from the β-fission which seemed to occur characteristically in CEI processes. The results obtained suggested that the formation of M^+. ions under matrix FAB conditions occurred mainly by CE reactions between the analytes M and matrix molecular ions B^+. and/or fragment ions b^+..     

On the risk of false positive identification using multiple ion monitoring in qualitative mass spectrometry: Large-scale intercomparisons with a comprehensive mass spectral library

Analysts involved in qualitative mass spectrometry have long debated the minimum data requirements for demonstrating that signals from an unknown sample are identical to those from a known compound. Often this process is carried out by comparing a few selected ions acquired by multiple ion monitoring (MIM), with due allowance for expected variability in response. In a few past experiments with electron-ionization mass spectrometry (EI-MS), the number of ions selected and the allowable variability in relative abundance were tested by comparing one spectrum against a library of mass spectra, where library spectra served to represent potential false positive signals in an analysis. We extended these experiments by carrying out large-scale intercomparisons between thousands of spectra and a library of one hundred thousand EI mass spectra. The results were analyzed to gain insights into the identification confidence associated with various numbers of selected ions. A new parameter was investigated for the first time, to take into account that a library spectrum with a different base peak than the search spectrum may still cause a false positive identification. The influence of peak correlation among the specific ions in all the library mass spectra was also studied. Our computations showed that (1) false positive identifications can result from similar compounds, or low-abundance peaks in unrelated compounds if the method calls for detection at very low levels; (2) a MIM method's identification confidence improves in a roughly continuous manner as more ions are monitored, about one order of magnitude for each additional ion selected; (3) full scan spectra still represent the best alternative, if instrument sensitivity is adequate. The use of large scale intercomparisons with a comprehensive library is the only way to provide direct evidence in support of these conclusions, which otherwise depend on the judgment and experience of individual analysts. There are implications for residue chemists who would rely on standardized confirmation criteria to assess the validity of a given confirmatory method. For example, standardized confirmation criteria should not be used in the absence of interference testing and rational selection of diagnostic ions.     

Automatic poisson peak harvesting for high throughput protein identification

High throughput identification of proteins by peptide mass fingerprinting requires an efficient means of picking peaks from mass spectra. Here, we report the development of a peak harvester to automatically pick monoisotopic peaks from spectra generated on matrix-assisted laser desorption/ionisation time of flight (MALDI-TOF) mass spectrometers. The peak harvester uses advanced mathematical morphology and watershed algorithms to first process spectra to stick representations. Subsequently, Poisson modelling is applied to determine which peak in an isotopically resolved group represents the monoisotopic mass of a peptide. We illustrate the features of the peak harvester with mass spectra of standard peptides, digests of gel-separated bovine serum albumin, and with Escherictia coli proteins prepared by two-dimensional polyacrylamide gel electrophoresis. In all cases, the peak harvester proved effective in its ability to pick similar monoisotopic peaks as an experienced human operator, and also proved effective in the identification of monoisotopic masses in cases where isotopic distributions of peptides were overlapping. The peak harvester can be operated in an interactive mode, or can be completely automated and linked through to peptide mass fingerprinting protein identification tools to achieve high throughput automated protein identification.     

Laser Desorption Ionization Versus Electrospray Ionization Mass Spectrometry: Applications in the Analysis of Cluster Anions

Mass spectra of transition metal carbonyl cluster anions were recorded using laser desorption ionization time-of-flight (LDI-TOF) and electrospray ionization (ESI) techniques. The LDI spectra generally contain peaks corresponding the intact cluster together with extensive CO loss fragments ions whereas the ESI spectra exhibit peaks corresponding the intact cluster together with few (if any) CO loss fragment ions. The parameters of both techniques can be modified to vary the extent of fragmentation. In all cases no fragmentation of the metal core is observed. Overall, ESI is a more informative method for the analysis of these types of cluster anions.     

Novel phosphoryl derivatization method for peptide sequencing by electrospray ionization mass spectrometry

A one-step phosphoryl derivatization method has been used in a peptide sequencing procedure for electrospray ionization tandem mass spectrometry (ESI-MS/MS). The sodiated derivatized peptides exhibit very simple dissociation patterns, in which two kinds of fragment ions, [b(n) + OH + Na]+ and [a(n) + Na]+, are formed. Since the amino acid residues are lost sequentially from the C-terminus, peptide sequences can be identified easily. The fragmentation efficiency of peptides increased as a result of the phosphorylation, and also provided peaks of useful intensity at lower m/z. A peptide with lysine at the C-terminus was derivatized and analyzed by ESI-MS/MS. Similar mass spectra, from which the sequence could be read out, were obtained. This is a novel derivatization method yielding neutral derivatives that should be suitable for peptide sequencing by LC/ESI-MS/MS.