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.     

A comparison of the peptide fragmentation obtained from a reflector matrix-assisted laser desorption-ionization time-of-flight and a tandem four sector mass spectrometer

The types, extent, and overall distribution of peptide fragmentation produced by matrix-assisted laser desorption-ionization-postsource decay (MALDI-PSD) on a reflector time-of-flight mass spectrometer were compared with those obtained from high and low energy collision-induced dissociation (CID) on a four-sector mass spectrometer and from liquid secondary ion mass spectrometry (LSIMS) ion source fragmentation and LSIMS metastable ion (MI) decomposition on a two-sector mass spectrometer. The model peptides studied had sequences and compositions that yielded predominantly either N- or C-terminal fragmentation from CID. For des-Arg¹ and des-Arg⁹ bradykinin (i.e., H-PPGFSPFR-OH and H-RP-PGFSPF-OH, respectively), the types of fragment ions and the extent to which each type is formed in both MALDI-PSD and low energy CID spectra are remarkably similar. This observation suggests that both methods deposit comparable internal energies (IE) into [M + H]⁺ precursor ions. The distribution of N-terminal, C-terminal, immonium, and internal fragmentation from MALDI-PSD spectra of des-Arg¹ and des-Arg⁹ bradykinin did not change dramatically with respect to the terminal arginine position, contrary to those from LSIMS MI decomposition, high and low energy CID spectra. This observation in combination with the prominent immonium, internal, and minus 17 fragment ion types in PSD indicates that the imparted IE from MALDI and the 14 µs of flight time may promote steady-state decomposition kinetics. Fragmentation distributions of MALDI-PSD spectra are also similar to those in LSIMS spectra. This implies that the distribution of protonation sites in [M + H]⁺ is comparable for both techniques.     

Deflection of laser-produced ions in laser-induced thermal-desorption/fourier-transform mass spectrometry for surface analysis

A method for deflecting ions, such as K⁺, produced outside a Fourier-transform mass spectrometer cell during laser-induced thermal desorption, is described. This technique has been shown to deflect laser-generated K and Ti ions from two Ti foil samples (biomedical implant model surfaces), yielding mass spectra of coadsorbed organic species. Further studies characterizing the laser desorption/deflection parameters have shown that ion deflection improves with higher deflection voltages and greater sample to Fourier-transform mass spectrometry cell separation. Higher laser power densities resulted in greater surface ion production; hence higher deflection voltages were necessary. A 6% increase in laser power necessitated a fourfold increase in deflection voltage for the Ti sample.     

Sequence and side-chain specific photofragment (193 nm) ions from protonated substance P by matrix-assisted laser desorption ionization time-of-flight mass spectrometry

Photodissociation at 193 nm (6. 43 eV) of the protonated substance-P, [M + H]⁺ ions, in a delayed extraction matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometer, is reported. The photofragment ion spectrum of substance P contains a complete series of a-type fragment ions and abundant side-chain cleavage ions. This article focuses on the utility of MALDI-TOF photodissociation for peptide sequencing.     

Analysis of high mass peptides using a novel matrix-assisted laser desorption/ionisation quadrupole ion trap time-of-flight mass spectrometer

A novel matrix-assisted laser desorption/ionisation quadrupole ion trap time-of-flight (MALDI QIT ToF) mass spectrometer has been used to analyse high mass peptide ions exceeding 2000 Da. Human adrenocorticotropic hormone (fragment 18-39) and oxidised bovine insulin chain B were utilised to evaluate the performance of the instrument both in MS and in MS/MS mode. Its ability to efficiently isolate ions and to fragment them using collisionally activated decomposition (CAD) has been demonstrated using mixtures diluted to the low-femtomole level on target. Additionally, multiple stage mass spectrometry (MS/MS/MS) provides a second-generation product ion spectrum in which new fragment ions are detected and new stretches of amino acids are identified.     

Formation of c1 fragment ions in collision-induced dissociation of glutamine-containing peptide ions: a tip for de novo sequencing

A c1 ion was observed with significant yield in the tandem mass (MS/MS) spectra of peptide ions containing glutamine as the second amino acid residue from the N-terminus. The c1 fragment was generated independently of the N-terminal residue of the peptide, but its abundance was strongly dependent on the side-chain identity. This ion is not a common fragmentation product in low-energy collision-induced dissociation of peptide ions, but it assists in identification of the first two amino acid residues, often difficult due to a low or absent signal from the heaviest y ion. A consecutive fragmentation mechanism is proposed, involving a b2 ion with a six-membered ring as an intermediate, to explain the exceptional stability of the c1 fragment ion. The utility of this information is discussed, especially in de novo sequencing of peptide ions.     

Reproducibility and extent of fragmentation of laser-desorbed ions : Relation to mechanism of desorption

The reproducibility of decomposition patterns of laser desorbed propyltriphenyl-phosphonium ions and (M + Na)⁺ ions of sucrose is evaluated. These compounds were chosen because they desorb by two different mechanisms commonly ascribed to “thermal” desorption. The phosphonium ions desorb directly whereas sucrose requires cationization with an alkali metal ion. Samples were either pipetted or electrosprayed onto both “smooth” and “rough” copper probe tips. The precision of two ratios of ion abundances are slightly worse for laser desorption of the phosphonium ions than for ions produced by electron ionization of toluene and 2,5-dibromotoluene. Little improvement was obtained by turning to electrospray as a means of depositing the sample. Worse precision was found for ion abundance ratios obtained from the laser desorption of (M + Na)⁺ of sucrose which hadbeen pipetted onto the probe. The precision was improved by using the electrospray deposition method. Fragmentation of the phosphonium ions was greater if desorption was from a “rough” probe tip whereas less decomposition of the (M + Na)⁺ of sucrose occurred with desorption from a “rough” tip. The trends in precision and extent of fragmentation are rationalized in terms of mechanisms of ion desorption. Chemical ionization of laser-desorbed neutral species was found to be impossible under the experimental conditions used.     

Characterization of sodiated glycerol phosphatidylcholine phospholipids by mass spectrometry

Fast atom bombardment mass spectrometry in the positive mode was used for the characterization of sodiated glycerol phosphatidylcholines. The relative abundance (RA) of the protonated species is similar to the RA of the sodiated molecular species. The sodiated fragment ion, [M + Na - 59](+), corresponding to the loss of trimethylamine, and other sodiated fragment ions, were also observed. The decomposition of the sodiated molecule is very similar for all the studied glycerol phosphatidylcholines, in which the most abundant ion corresponds to a neutral loss of 59 Da. Upon collision-induced dissociation (CID) of the [M + Na](+) ion informative ions are formed by the losses of the fatty acids in the sn-1 and sn-2 positions. Other major fragment ions of the sodiated molecule result from loss of non-sodiated and sodiated choline phosphate, [M + Na - 183](+), [M + Na - 184](+.) and [M + Na - 205](+), respectively. The main CID fragmentation pathway of the [M + Na - 59](+) ion yields the [M + Na - 183](+) ion, also observed in the CID spectra of the [M + Na](+) molecular ion. Other major fragment ions are [M + Na - 205](+) and the fragment ion at m/z 147. Collisional activation of [M + Na - 205](+) results in charge site remote fragmentation of both fatty acid alkyl chains. The terminal ions of these series of charge remote fragmentations result from loss of part of the R(1) or R(2) alkyl chain. Other major informative ions correspond to acylium ions.     

Role of the UV absorber as a matrix in matrix‐assisted laser desorption/ionization mass spectrometric analysis of a mixture of a UV absorber and a stabilizer

A mixture of a UV absorber (Tinuvin 234 or Tinuvin 329) and a UV stabilizer (Tinuvin 770) was analyzed using matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) without any matrix. Fragmentation patterns of the UV absorbers and stabilizer were also investigated. The mass spectra showed the [M+H]⁺ ions and some fragment ions. Tinuvin 234, Tinuvin 329, and Tinuvin 770 generated three (m/z 119, 370, 432), one (m/z 252), and two (m/z 124 and 140) fragment ions, repectively. These fragment ions can be used to identify the chemical structures of the UV absorbers and stabilizer. Since the UV absorber performed a role as the matrix, the ion abundance of the UV stabilizer was enhanced by mixing with the UV absorber. When organic materials extracted from polypropylene (PP) containing the UV absorber and stabilizer were directly analyzed using MALDI‐MS without any matrix, the protonated molecule of the UV stabilizer was detected in abundance but the product ions of the UV absorber were not observed. When 2,5‐dihydroxybenzoic acid was used as a matrix, the protonated molecule of the UV absorber was observed. Copyright © 2010 John Wiley & Sons, Ltd.     

Matrix-assisted laser desorption/ionization time-of-flight and nano-electrospray ionization ion trap mass spectrometric characterization of 1-cyano-2-substituted-benz[f]isoindole derivatives of peptides for fluorescence detection

A series of hexa- to decapeptides (molecular mass range 800-1200) were labeled with naphthalene-2,3-dicarboxaldehyde, which preferentially reacts with the primary amino groups of a peptide. A highly stable peptide conjugate is formed, which allows selective analysis by fluorescence at excitation and emission wavelengths of 420 and 490 nm, respectively. After removal of unreacted compounds, the peptide conjugates were characterized by matrix-assisted laser desorption/ionization (MALDI) time-of-flight and nano-electrospray ionization (ESI) ion trap mass spectrometry. They readily form both [M + H]+ ions by MALDI and both [M + H]+ and [M + 2H]2+ ions by ESI. Furthermore, the fragmentation behavior of the N-terminally tagged peptides, exhibiting an uncharged N-terminus, was investigated applying post-source decay fragmentation with a curved field reflector and collision-induced dissociation with a quadrupole ion trap. Fragmentation is dominated in both cases by series of a-, b- and y-type ions and [M + H - HCN]+ ions. Peptide bonds adjacent to the fluorescence label were less susceptible to cleavage than the bonds of the non-derivatized peptide ions. In general, the resulting fragment ion patterns were less complex than those of the underivatized peptides.     

The observation of broad metastable ion peaks in the surface-induced dissociation mass spectra of protonated and cationated peptides

The product ion mass spectra of protonated and cationated peptides of relative molecular mass (RMM) 555–574 Da have been obtained by surface-induced dissociation of MH⁺ and [M + Cat]⁻ ions in a four-sector tandem mass spectrometer equipped with a specially designed collision cell. A linked scan of the electric and magnetic sector field strengths of the second mass spectrometer was used to transmit the fragment ions arising from collisions with a stainless steel surface. The resulting mass spectra contained broad metastable ion peaks produced by the dissociation of MH⁺ and [M + Cat]⁺ ions before the second magnetic sector, in the fourth field-free region of the instrument.     

Intrinsic acidity and basicity of neutral and ionic species of some polyether-ester compounds as shown by mass spectrometry

The mass spectrometric behaviour of benzo-[o]-1,4,7,10,13-pentaoxacycloheptadecane-14,17-dione (1), 3,6,9,12,15-pentaoxabicyclo[15.3.1]heneicosa-1(21),17,19-triene-2,16-dione (2) and 1,15-dioxo-2,5,8,11,14-pentaoxa[15](1,4)benzenophane (3) has been studied in detail with the aid of linked scans, mass-analysed ion kinetic energy spectra, collisionally activated decomposition experiments, exact mass measurements and different ionization techniques (electron impact, positive and negative ion chemical ionization, charge exchange, electron attachment). The very low abundance of molecular ions and the presence of abundant [M + H]⁺ and [M – H]⁺ ions under electron impact conditions indicate the presence of acidic and basic sites in the molecular ions and neutral molecules, respectively.     

Mass spectrometric methods for distinguishing structural isomers of glutathione conjugates of estrone and estradiol

Collisionally activated decompositions (CAD) of [M+H]⁺ ions from two sets (estrone and estradiol) of three isomeric glutathione (GSH) conjugates were studied by using five tandem mass spectrometric methods: (1) low energy (LE) CAD in an ion trap, (2) LE CAD in a triple quadrupole, (3) electrospray ionization (ESI)-source CAD in a tandem four sector, (4) high energy (HE) CAD of both ESI-produced and fast-atom bombardment (FAB)-produced ions in a tandem four-sector mass spectrometer, and (5) metastable-ion decompositions of FAB-produced ions. Four types of fragment ions are produced. The first type, formed from cleavage of the peptide backbone, gives rise to modified b₂, modified y₂, y₂, and b₁ ions. These fragments are observed with all the methods and show that the catechol estrogen attachment is at the cysteine moiety of the GSH. Internal fragment ions are the second type, and they also support that the modification is at cysteine. The third type involves fragmentation of the C–S bond to give an ion containing the steroid bonded to the sulfur. The fourth type of fragment ion is similar to the third but involves oxidation of the steroid ring and reduction of the GSH moiety; it is the most isomer specific of the four. The isomer-specific ions are of relatively low abundance in the product-ion spectra taken on the triple quadrupole and ion trap, but their abundances can be improved by increasing the collision energy. ESI source-CAD and the HE-CAD spectra of the isomers are the most distinctive because abundant product ions of all four types are seen in a single spectrum.     

Efficiency of collisionally-activated dissociation and 193-nm photodissociation of peptide ions in fourier transform mass spectrometry

For tandem mass spectrometry, the Fourier transform instrument exhibits advantages for the use of collisionally-activated dissociation (CAD). The CAD energy deposited in larger ions can be greatly increased by extending the collision time to as much as 120 s, and the efficiency of trapping and measuring CAD product ions is many times greater than that found for triple-quadrupole or magnetic sector instruments, although the increased pressure from the collision gas is an offsetting disadvantage. A novel system that uses the same laser for photodesorption of ions and their subsequent photodissociation can produce complete dissociation of larger oligopeptide ions and unusually abundant fragment ions. In comparison to CAD, much more internal energy can be deposited in the primary ions using 193-nm photons, sufficient to dissociate peptide ions of m/z > 2000. Mass spectra closely resembling ion photodissociation spectra can also be obtained by' neutral photodissociation (193-nm laser irradiation of the sample) followed by ion photodesorption.     

Electron-impact induced fragmentation of substituted pyrazolone azomethine dyes

The mass spectra of 18 substituted 1-(p-R₂-phyeyl)-3-R₃-4-(p-R₁-phenylimino)-2-pyrazolin-5-ones have been obtained. The major cleavages under electron bombardment take place in the pyrazoline nucleus, producing four major fragment ions whose abundance is related primarily to the electronic properties of R₁. These ions appear to be produced from the molecular ion via an intermediate formed by the loss of CO. In most instances metastable ions are observed for the overall decomposition of the molecular ion to the four major fragment ions. A mechanism is proposed for these cleavages.     

Charge remote fragmentation of peptides following attachment of a fixed positive charge: A matrix-assisted laser desorption/ionization postsource decay study

A fixed positive charge can be placed at the N-terminus of a peptide by addition of a tris[(2,4,6-trimethoxyphenyl)phosphonium]acetyl group. The usefulness of these charged derivatives has been demonstrated in fast atom bombardment mass spectrometry and in matrix-assisted laser desorption/ionization mass spectrometry. After ion formation and acceleration, these derivatized peptide ions dissociate and their fragment ions can be analyzed in a postsource decay experiment by using a time-of-flight mass spectrometer. The matrix-assisted laser desorption/ionization-postsource decay spectra are very different from what may be expected based on fragment ions observed from protonated peptide molecules. Cleavage of CHR-C(O) bonds dominates to form a series of a type ions. Mechanistic possibilities are evaluated. When aspartic acid residues are encountered, the chemistry radically changes. This appears to be due to the formation of geometrically accessible intermediates that can dissociate via low energy processes. This chargeremote chemistry parallels that for much simpler systems, resulting in spectra that are very easy to interpret.     

Fragmentation chemistry of [M + Cu]+ peptide ions containing an N-terminal arginine

[M + Cu]+ peptide ions formed by matrix-assisted laser desorption/ionization from direct desorption off a copper sample stage have sufficient internal energy to undergo metastable ion dissociation in a time-of-flight mass spectrometer. On the basis of fragmentation chemistry of peptides containing an N-terminal arginine, we propose the primary Cu+ ion binding site is the N-terminal arginine with Cu+ binding to the guanidine group of arginine and the N-terminal amine. The principal decay products of [M + Cu]+ peptide ions containing an N-terminal arginine are [a(n) + Cu - H]+ and [b(n) + Cu - H]+ fragments. We show evidence to suggest that [a(n) + Cu - H]+ fragment ions are formed by elimination of CO from [b(n) + Cu - H]+ ions and by direct backbone cleavage. We conclude that Cu+ ionizes the peptide by attaching to the N-terminal arginine residue; however, fragmentation occurs remote from the Cu+ ion attachment site involving metal ion promoted deprotonation to generate a new site of protonation. That is, the fragmentation reactions of [M + Cu]+ ions can be described in terms of a "mobile proton" model. Furthermore, proline residues that are adjacent to the N-terminal arginine do not inhibit formation of [b(n) + Cu - H]+ ion, whereas proline residues that are distant to the charge carrying arginine inhibit formation of [b(n) + Cu - H]+ ions. An unusual fragment ion, [c(n) + Cu + H]+, is also observed for peptides containing lysine, glutamine, or asparagine in close proximity to the Cu+ carrying N-terminal arginine. Mechanisms for formation of this fragment ion are also proposed.     

A collinear tandem time-of-flight mass spectrometer for infrared photodissociation spectroscopy of mass-selected ions

An apparatus based on collinear tandem time-of-flight mass spectrometer has been designed for the measurement of infrared photodissociation spectroscopy of mass-selected ions in the gas phase.The ions from a pulsed laser vaporization supersonic ion source are skimmed and mass separated by a Wiley-McLaren time-of-flight mass spectrometer.The ion of interest is mass selected,decelerated and dissociated by a tunable IR laser.The fragment and parent ions are reaccelerated and mass analyzed by the second time-of-flight mass spectrometer.A simple new assembly integrated with mass gate,deceleration and reacceleration ion optics was designed,which allows us to measure the infrared spectra of mass selected ions with high sensitivity and easy timing synchronization.     

"De novo" peptide sequencing by MALDI-quadrupole-ion trap mass spectrometry: a preliminary study

Collision-induced dissociation of singly charged peptide ions produced by resonant excitation in a matrix-assisted laser desorption/ionization (MALDI) ion trap mass spectrometer yields relatively low complexity MS/MS spectra that exhibit highly preferential fragmentation, typically occurring adjacent to aspartyl, glutamyl, and prolyl residues. Although these spectra have proven to be of considerable utility for database-driven protein identification, they have generally been considered to contain insufficient information to be useful for extensive de novo sequencing. Here, we report a procedure for de novo sequencing of peptides that uses MS/MS data generated by an in-house assembled MALDI-quadrupole-ion trap mass spectrometer (Krutchinsky, Kalkum, and Chait Anal. Chem. 2001, 73, 5066-5077). Peptide sequences of up 14 amino acid residues in length have been deduced from digests of proteins separated by SDS-PAGE. Key to the success of the current procedure is an ability to obtain MS/MS spectra with high signal-to-noise ratios and to efficiently detect relatively low abundance fragment ions that result from the less favorable fragmentation pathways. The high signal-to-noise ratio yields sufficiently accurate mass differences to allow unambiguous amino acid sequence assignments (with a few exceptions), and the efficient detection of low abundance fragment ions allows continuous reads through moderately long stretches of sequence. Finally, we show how the aforementioned preferential cleavage property of singly charged ions can be used to facilitate the de novo sequencing process.     

In-source decay and fragmentation characteristics of peptides using 5-aminosalicylic acid as a matrix in matrix-assisted laser desorption/ionization mass spectrometry

The use of 5-aminosalicylic acid (5-ASA) as a new matrix for in-source decay (ISD) of peptides including mono- and di-phosphorylated peptides in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) is described. The use of 5-ASA in MALDI-ISD has been evaluated from several standpoints: hydrogen-donating ability, the outstanding sharpness of molecular and fragment ion peaks, and the presence of interference peaks such as metastable peaks and multiply charged ions. The hydrogen-donating ability of several matrices such as α-cyano-4-hydroxycinnamic acid (CHCA), 2,5-dihydroxybenzoic acid (2,5-DHB), 1,5-diaminonaphthalene (1,5-DAN), sinapinic acid (SA), and 5-ASA was evaluated by using the peak abundance of a reduction product [M + 2H + H]⁺ to that of non-reduced protonated molecule [M + H]⁺ of the cyclic peptide vasopressin which contains a disulfide bond (S-S). The order of hydrogendonating ability was 1,5-DAN > 5-ASA > 2,5-DHB > SA = CHCA. The chemicals 1,5-DAN and 5-ASA in particular can be classified as reductive matrices. 5-ASA gave peaks with higher sharpness for protonated molecules and fragment ions than other matrices and did not give any interference peaks such as multiply-protonated ions and metastable ions in the ISD mass spectra of the peptides used. Particularly, 1,5-DAN and 5-ASA gave very little metastable peaks. This indicates that 1,5-DAN and 5-ASA are more “cool” than other matrices. The 1,5-DAN and 5-ASA can therefore be termed “reductive cool” matrix. Further, it was confirmed that ISD phenomena such as N-Cα bond cleavage and reduction of S-S bond is a single event in the ion source. The characteristic fragmentations, which form a− and (a + 2)-series ions, [M + H − 15]⁺, [M + H − 28]⁺, and [M + H − 44]⁺ ions in the MALDI-ISD are described.