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.     

The nature of collision-induced dissociation processes of doubly protonated peptides: comparative study for the future use of matrix-assisted laser desorption/ionization on a hybrid quadrupole time-of-flight mass spectrometer in proteomics.

Comparative MS/MS studies of singly and doubly charged electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) precursor peptide ions are described. The spectra from these experiments have been evaluated with particular emphasis on the data quality for subsequent data processing and protein/amino acid sequence identification. It is shown that, once peptide ions are formed by ESI or MALDI, their charge state, as well as the collision energy, is the main parameter determining the quality of collision-induced dissociation (CID) MS/MS fragmentation spectra of a given peptide. CID-MS/MS spectra of singly charged peptides obtained on a hybrid quadrupole orthogonal time-of-flight mass spectrometer resemble very closely spectra obtained by matrix-assisted laser desorption/ionization post-source decay time-of-flight mass spectrometry (MALDI-PSD-TOFMS). On the other hand, comparison of CID-MS/MS spectra of either singly or doubly charged ion species shows no dependence on whether ions have been formed by ESI or MALDI. This observation confirms that, at the time of precursor ion selection, further mass analysis is effectively decoupled from the desorption/ionization event. Since MALDI ions are predominantly formed as singly charged species and ESI ions as doubly charged, the associated difference in the spectral quality of MS/MS spectra as described here imposes direct consequences on data processing, database searching using ion fragmentation data, and de novo sequencing when ionization techniques are changed.     

Simultaneous measurement of flight time and energy of large matrix-assisted laser desorption ionization ions with a superconducting tunnel junction detector

We evaluated a cryogenically cooled superconducting Nb-Al₂O₃-Nb tunnel junction (STJ) for use as a molecular ion detector in a matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometer. The STJ responds to ion energy and theoretically should detect large molecular ions with a velocity-independent efficiency approaching 100%. The STJ detector produces pulses whose heights are approximately proportional to ion energy, thus the height of a pulse generated by the impact of a doubly charged ion is about twice the height of a singly charged ion pulse. Measurements were performed by bombarding the STJ with human serum albumin (HSA) (66,000 Da) and immunoglobulin (150,000 Da) ions. We demonstrate that pulse height analysis of STJ signals provides a way to distinguish with good discrimination HSA⁺ from 2HSA²⁺, whose flight times are coincident. The rise time of STJ detector pulses allows ion flight times to be determined with a precision better than 200 ns, which is a value smaller than the flight time variation typically observed for large isobaric MALDI ions detected with conventional microchannel plate (MCP) detectors. Deflection plates in the flight tube of the mass spectrometer provided a way to aim ions alternatively at a MCP ion detector.     

Automated assignment of ionization states in broad‐mass matrix‐assisted laser desorption/ionization spectra of protein mixtures

A computational technique is presented for the automated assignment of the multiple charge and multimer states (ionization states) in the time‐of‐flight (TOF) domain for matrix‐assisted laser desorption/ionization (MALDI) spectra. Examples of the application of this technique include an improved, automatic calibration over the 2 to 70 kDa mass range and a reduced data redundancy after reconstruction of the molecular spectrum of only singly charged monomers. This method builds on our previously reported enhancement of broad‐mass signal detection, and includes two steps: (1) an automated correction of the instrumental acquisition initial time delay, and (2) a recursive TOF detection of multiple charge states and singly charged multimers of molecular [MH]⁺ ions over the entire record range, based on MALDI methods. The technique is tested using calibration mixtures and pooled serum quality control samples acquired along with clinical study data. The described automated procedure improves the analysis and dimension reduction of MS data for comparative proteomics applications. Copyright © 2009 John Wiley & Sons, Ltd.     

Direct differentiation of A‐ring single attachment versus A‐ and D‐ring double attachment of phycoerythrobilin chromophores to phycobiliproteins using MALDI mass spectrometry

Bilin chromophore attachment to phycobiliproteins is an enzyme‐catalyzed post‐translational modification process. Bilin‐lyases attach a bilin chromophore to their cognate protein through a thioether bond between the chromophore and a cysteine moiety. Bilin chromophores are attached to their phycobiliproteins through the 3¹ carbon of the bilin. Double attachment may also occur, and in this case, carbons 3¹ and 18¹ of the bilin are both forming covalent linkages to cysteine moieties. There is a mass spectrometric limitation when examining tryptic peptides containing two (or more) cysteines if one seeks to ascertain whether chromopeptides are singly or doubly attached. The problem is that singly and doubly attached chromopeptides appear at the same m/z value; thus, up until the present, only NMR analysis has been successful at determining whether the chromophore is singly or doubly attached. We report in this work a new, fast and accurate method for discriminating singly from doubly attached chromophores using MALDI‐TOF mass spectrometry. This method was developed from mass spectral analysis of chromopeptides that had undergone in vitro or in vivo attachment of bilin chromophores to phycobiliproteins. Distinction is based on a characteristic neutral loss that appears in the MALDI‐TOF mass spectrum only when the bilin is singly attached. Copyright © 2013 John Wiley & Sons, Ltd.     

Observation of doubly charged mercury cluster ions Hg n 2+, n=1-10 using secondary ion mass spectrometry

Mass spectra of doubly charged mercury clusters (m/z=30-1065) were investigated by secondary ion mass spectrometry. Positively charged ions were generated from an amalgam of mercury and silver by bombardment with a xenon ion beam and mass analysis by a grand-scale sector type mass spectrometer. Hg _n ²⁺, n=1-10 and Hg _n +, n =1- 5 were observed. Some doubly charged mercury clusters, (Hg _n ²⁺) survived at least for 0.1 ms.     

Fragmentation of organic ions bearing fixed multiple charges observed in MALDI MS

Matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI TOF MS) was used to analyze a series of synthetic organic ions bearing fixed multiple charges. Despite the multiple intrinsic charges, only singly charged ions were recorded in each case. In addition to the pseudo‐molecular ions formed by counterion adduction, deprotonation and electron capture, a number of fragment ions were also observed. Charge splitting by fragmentation was found to be a viable route for charge reduction leading to the formation of the observed singly charged fragment ions. Unlike multivalent metal ions, organic ions can rearrange and/or fragment during charge reduction. This fragmentation process will evidently complicate the interpretation of the MALDI MS spectrum. Because MALDI MS is usually considered as a soft ionization technique, the fragment ion peaks can easily be erroneously interpreted as impurities. Therefore, the awareness and understanding of the underlying MALDI‐induced fragmentation pathways is essential for a proper interpretation of the corresponding mass spectra. Due to the fragment ions generated during charge reduction, special care should be taken in the MALDI MS analysis of multiply charged ions. In this work, the possible mechanisms by which the organic ions bearing fixed multiple charges fragment are investigated. With an improved understanding of the fragmentation mechanisms, MALDI TOF MS should still be a useful technique for the characterization of organic ions with fixed multiple charges.     

Tandem mobility mass spectrometry study of electrosprayed tetraheptyl ammonium bromide clusters

Multiply charged electrospray ions from concentrated solutions of Heptyl4N+Br- (designated A+B- hereafter) in formamide are analyzed mass spectrometrically (MS) following mobility selection in ambient air in a differential mobility analyzer (DMA). Most of the sharp mobility peaks seen are identified as (AB)(n)A+ clusters, with 0 < or = n < ot = 5. One anomalously abundant and mobile ion is identified as NH4+(AB)4. Six ions in the (AB)n(A+)2 series are also identified, completing and correcting earlier mobility data for singly and doubly charged ions up to masses of almost 9000 Da. The more mobile of two broad humps seen in the mobility spectrum includes m/z values approximately from 2500 up to 12,000 Da. It is formed primarily by multiply charged (AB)n(A+)z clusters with multiple ammonium bromide adducts. Because of overlapping of many peaks of different m/z and charge state z, only a few individual species can be identified by MS alone in this highly congested region. However, the spectral simplification brought about by mobility selection upstream of the MS reveals a series of broad modulations in m/z space, with all ions resolved in the second, third, ...sixth modulation being in charge states z = 2, 3, ...6, respectively. Extrapolation of this trend beyond the sixth wave fixes the ion charge state (in some cases up to z = 15) and mass (beyond m = 175,000 u). This wavy structure had been previously observed and explained in terms of ion evaporation kinetics from volatile drops, though without mass identification. All observations indicate that the clusters are formed as charged residues, but their charge state is fixed by the Iribarne-Thomson ion evaporation mechanism. Consequently, the measured curve of cluster diameter versus z yields the two parameters governing ion evaporation kinetics. Clusters with z > 1 and electrical mobility Z > 0.495 cm2/V/s are metastable and evaporate a singly charged cluster, probably (AB)2A+, between the DMA and the MS. Plotting the electrical mobilities Z of the clusters in the form (z/Z)1/2 versus m(1/3) (both proportional to cluster diameter) collapse the data for all cluster sizes and charge states into one single straight line for Z below 0.495 cm2/V/s. This linear relation reveals a uniform apparent cluster density of 0.935 g/cm3 and an effective hard-sphere diameter of the air molecules of 0.44 nm. An anomalous mobility increase is observed at diameters below 3 nm.     

Phosphorylated serine and threonine residues promote site‐specific fragmentation of singly charged,arginine‐containing peptide ions

In order to investigate gas‐phase fragmentation reactions of phosphorylated peptide ions, matrix‐assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) tandem mass (MS/MS) spectra were recorded from synthetic phosphopeptides and from phosphopeptides isolated from natural sources. MALDI‐TOF/TOF (TOF: time‐of‐flight) spectra of synthetic arginine‐containing phosphopeptides revealed a significant increase of y ions resulting from bond cleavages on the C‐terminal side of phosphothreonine or phosphoserine. The same effect was found in ESI‐MS/MS spectra recorded from the singly charged but not from the doubly charged ions of these phosphopeptides. ESI‐MS/MS spectra of doubly charged phosphopeptides containing two arginine residues support the following general fragmentation rule: Increased amide bond cleavage on the C‐terminal side of phosphorylated serines or threonines mainly occurs in peptide ions which do not contain mobile protons. In MALDI‐TOF/TOF spectra of phosphopeptides displaying N‐terminal fragment ions, abundant b–H₃PO₄ ions resulting from the enhanced dissociation of the pSer/pThr–X bond were detected (X denotes amino acids). Cleavages at phosphoamino acids were found to be particularly predominant in spectra of phosphopeptides containing pSer/pThr–Pro bonds. A quantitative evaluation of a larger set of MALDI‐TOF/TOF spectra recorded from phosphopeptides indicated that phosphoserine residues in arginine‐containing peptides increase the signal intensities of the respective y ions by almost a factor of 3. A less pronounced cleavage‐enhancing effect was observed in some lysine‐containing phosphopeptides without arginine. The proposed peptide fragmentation pathways involve a nucleophilic attack by phosphate oxygen on the carbon center of the peptide backbone amide, which eventually leads to cleavage of the amide bond. Copyright © 2009 John Wiley & Sons, Ltd.     

Ion mobility augments the utility of mass spectrometry in the identification of human hemoglobin variants

The global dispersion of hemoglobin variants through population migration has precipitated a need for their identification. A particularly effective mass spectrometry (MS)-based procedure involves analysis of the intact globin chains in diluted blood to detect the variant through mass anomalies, followed by location of the variant amino acid residue by direct analysis of the enzymatically digested globins. Here we demonstrate the use of ion mobility separation in combination with this MS procedure to reduce mass spectral complexity. In one example, the doubly charged tryptic peptide from a low abundance variant (4%) occurred at the same m/z value as a singly and a doubly charged interfering ion. In another example, the singly charged tryptic peptide from an alpha-chain variant (26%) occurred at the same m/z value as a doubly charged interfering ion. Ion mobility was used to separate the variant ions from the interfering ions, thus allowing the variant peptides to be observed and sequenced by tandem mass spectrometry.     

COMPLX: a computer algorithm for the detection of protein-ligand and other macromolecular complexes in mass spectra

A new algorithm has been designed and tested to identify protein, or any other macromolecular, complexes that have been widely reported in mass spectral data. The program takes advantage of the appearance of multiply charged ions that are common to both electrospray ionization and, to a lesser extent, matrix-assisted laser desorption/ionization (MALDI) mass spectra. The algorithm, known as COMPLX for the COMposition of Protein-Ligand compleXes, is capable of identifying complexes for any protein or macromolecule with a binding partner of molecular mass up to 100 000 Da. It does so by identifying ion pairs present in a mass spectrum that, when they share a common charge, have an m/z value difference that is an integer fraction of a ligand or binding partner molecular mass. Several additional criteria must be met in order for the result to be ranked in the output file including that all m/z values for ions of the protein or complex have progressively lower values as their assigned charge increases, the difference between the m/z values for adjacent charge states (z, z + 1) decrease as the assigned charge state increases, and the ratio of any two m/z values assigned to a protein or complex is equal to the inverse ratio of their charge. The entries that satisfy these criteria are then ranked according to the appearance of ions in the mass spectrum associated with the binding partner, the length of a continuous series of charges across any set of ions for a protein and complex and the lowest error recorded for the molecular mass of the ligand or binding partner. A diverse range of hypothetical and experimental mass spectral data were used to implement and test the program, including those recorded for antibody-peptide, protein-peptide and protein-heme complexes. Spectra of increasing complexity, in terms of the number of ions input, were also successfully analysed in which the number of input m/z values far exceeds the few associated with a macromolecular complex. Thus the program will be of value in a future goal of proteomics, where mass spectrometry already plays a central role, for the direct analysis of protein and other associations within biological extracts.     

双电荷离子[C12H12N2O]2+和[C12H12N2S]2+的气相单分子分解反应研究

The unimolecular dissociation reactions of doubly charged ions were reported,which resulted from a tandem mass spectrometer and a reversed geometry double focusing mass spectrometer by electron impact.Mass analyzed ion kinetic energy spectrometry(MIKES) was used to obtain the kinetic energy releases in charge separation reactions of doubly charged ions.The intercharge distances between the two charges at transition states can be calculated from the kinetic energy releases.Transition structures of unimolecular dissociation reactions were infered from MIKES and MS/MS.     

双电荷离子［C_（12）H_（12）N_2O］~（2＋）和［C_（12）H_（12

双电荷离子［Ｃ_（１２）Ｈ_（１２）Ｎ_２Ｏ］~（２＋）和［Ｃ_（１２）Ｈ_（１２）Ｎ_２Ｓ］~（２＋）的气相单分子分解反应研究任达，贾维平，李智立，刘淑莹（中国科学院长春应用化学研究所，长春，１３００２２）关键词双电荷离子，质量分析离子动能谱，串联质谱，４...     

Mass spectrometric characterization of the zein protein composition in maize flour extracts upon protein separation by SDS‐PAGE and 2D gel electrophoresis

Highly homogenous α zein protein was isolated from maize kernels in an environment‐friendly process using 95% ethanol as solvent. Due to the polyploidy and genetic polymorphism of the plant source, the application of high resolution separation methods in conjunction with precise analytical methods, such as MALDI‐TOF‐MS, is required to accurately estimate homogeneity of products that contain natural zein protein. The α zein protein product revealed two main bands in SDS‐PAGE analysis, one at 25 kDa and other at 20 kDa apparent molecular mass. Yet, high resolution 2DE revealed approximately five protein spot groups in each row, the first at ca. 25 kDa and the second at ca. 20 kDa. Peptide mass fingerprinting data of the proteins in the two dominant SDS‐PAGE bands matched to 30 amino acid sequence entries out of 102 non‐redundant data base entries. MALDI‐TOF‐MS peptide mapping of the proteins from all spots indicated the presence of only α zein proteins. The most prominent ion signals in the MALDI mass spectra of the protein mixture of the 25 kDa SDS gel band after in‐gel digestion were found at m/z 1272.6 and m/z 2009.1, and the most prominent ion signals of the protein mixture of the 20 kDa band after in‐gel digestion were recorded at m/z 1083.5 and m/z 1691.8. These ion signals have been found typical for α zein proteins and may serve as marker ion signals which upon chymotryptic digestion reliably indicate the presence of α zein protein in two hybrid corn products.     

2,5—双（4—羟基苯亚甲基）环戊酮中双电荷离子存在的串联质谱证据

应用串联质谱的碰撞诱导解离和联动扫描技术，研究了２，５－双（４－羟基苯亚甲基）环戊酮的质谱解离特征，提供了双电荷离子存在的实验证据。进一步对双电荷离子（ｍ／ｚ１４６）的碰撞诱导解离碎裂进行了讨论。     

Charge-induced unfolding of multiply charged polyethylene glycol ions

The electrical mobility of mass-selected single poly(ethylene glycol) (PEG) chains of mass m (<14 kDalton) and charge state z (+1 to +5) reveals a near-spherical shape above a critical mass m(z) approximately z(2). The abrupt unfolding observed at m < m(z) shows that the polymer molecules behave as liquid drops upon reaching the Rayleigh limit, with an apparent surface energy of 0.026 N/m at ion diameters from 1.7 to 3.2 nm. Other nonspherical shape families with structures independent of charge, and with charge-dependent stability domains, are observed. Highly charged ions adopt approximately linear highly stretched configurations where the mobility depends only on m/z, independently of z. An operational definition of the surface energy of a single long chain molecule that is computable and agrees with the measured surface energy is provided.     

Identification of Enterobacteriaceae bacteria by direct matrix-assisted laser desorptiom/ionization mass spectrometric analysis of whole cells.

Several members of Enterobacteriaceas were analyzed by matrix-assisted laser desorption ionization (MALDI) time-of-flight mass spectrometry (TOFMS). Characteristic mass spectral peaks and patterns were observed in the mass range of 2 to 20 kDa. The mass peaks reported to be reproducibly observed by previous researchers, which were claimed to serve as species/strain-specific biomarkers, are consistently observed in our current study. Despite the high degree of similarity found in the MALDI mass spectra within the enteric bacteria, minor yet notable differences existed to allow their differentiation. Five spectral peaks at m/z 4364, 5380, 6384, 6856, and 9540, generated reproducibly for each genus studied here, are assigned as family-specific biomarkers for the Family Enterobacteriaceae. The mass peaks at m/z 7324, 7724, 9136, and 9253 are assigned as genus-specific biomarkers for Salmonella. Some unique biomarkers characterizing the species and strains of E. coli are also presented.     

基质中加入少量盐类改善MALDI-TOF MS的谱图信噪比

自20世纪80年代发明基质辅助激光解吸电离(Matrix assisted laser desorption ionization,MALDI)质谱以来,该技术已在生物分子分析方面得到了广泛应用．作为一种离子化方法,MALDI具有灵敏度高,对样品要求低,能耐高浓度盐和缓冲剂等优点．测定过程中使用合适的基质不仅能提高测试灵敏度和分辨率,还能扩增测试样品的种类。     

Quantitative mapping of glycoprotein micro‐heterogeneity and macro‐heterogeneity: an evaluation of mass spectrometry signal strengths using synthetic peptides and glycopeptides

Mass spectrometry (MS) is used to quantify the relative distribution of glycans attached to particular protein glycosylation sites (micro‐heterogeneity) and evaluate the molar site occupancy (macro‐heterogeneity) in glycoproteomics. However, the accuracy of MS for such quantitative measurements remains to be clarified. As a key step towards this goal, a panel of related tryptic peptides with and without complex, biantennary, disialylated N‐glycans was chemically synthesised by solid‐phase peptide synthesis. Peptides mimicking those resulting from enzymatic deglycosylation using PNGase F/A and endo D/F/H were synthetically produced, carrying aspartic acid and N‐acetylglucosamine‐linked asparagine residues, respectively, at the glycosylation site. The MS ionisation/detection strengths of these pure, well‐defined and quantified compounds were investigated using various MS ionisation techniques and mass analysers (ESI‐IT, ESI‐Q‐TOF, MALDI‐TOF, ESI/MALDI‐FT‐ICR‐MS). Depending on the ion source/mass analyser, glycopeptides carrying complex‐type N‐glycans exhibited clearly lower signal strengths (10–50% of an unglycosylated peptide) when equimolar amounts were analysed. Less ionisation/detection bias was observed when the glycopeptides were analysed by nano‐ESI and medium‐pressure MALDI. The position of the glycosylation site within the tryptic peptides also influenced the signal response, in particular if detected as singly or doubly charged signals. This is the first study to systematically and quantitatively address and determine MS glycopeptide ionisation/detection strengths to evaluate glycoprotein micro‐heterogeneity and macro‐heterogeneity by label‐free approaches. These data form a much needed knowledge base for accurate quantitative glycoproteomics. Copyright © 2013 John Wiley & Sons, Ltd.     

Precision profiling and identification of human serum peptides using Fourier transform ion cyclotron resonance mass spectrometry

Many biomarker discovery studies are based on matrix-assisted laser desorption/ionisation (MALDI) peptide profiles. In this study, 96 human serum samples were analysed on a Bruker solariX(TM) MALDI Fourier transform ion cyclotron resonance (FTICR) system equipped with a 15 tesla magnet. Isotopically resolved peptides were observed in ultrahigh resolution FTICR profiles up to m/z 6500 with mass measurement errors (MMEs) of previously identified peptides at a sub-ppm level. For comparison with our previous platform for peptide profile mass analysis (i.e. Ultraflex II) the corresponding time-of-flight (TOF) spectra were obtained with isotopically resolved peptides up to m/z 3500. The FTICR and TOF systems performed rather similar with respect to the repeatability of the signal intensities. However, the mass measurement precision improved at least 10-fold in ultrahigh resolution data and thus simplified spectral alignment necessary for robust and quantitatively precise comparisons of profiles in large-scale clinical studies. From each single MALDI-FTICR spectrum an m/z-list was obtained with sub-ppm precision for all different species, which is beneficial for identification purposes and interlaboratory comparisons. Furthermore, the FTICR system allowed new peptide identifications from collision-induced dissociation (CID) spectra using direct infusion of reversed-phase (RP) C(18)-fractionated serum samples on an electrospray ionisation (ESI) source.