Method to compare collision-induced dissociation spectra of peptides: potential for library searching and subtractive analysis

We report the development of a method to compare collision-induced dissociation (CID) spectra of peptides. This method employs a cross-correlation analysis of a CID spectrum to a reference spectrum and normalizes the cross-correlation score to the autocorrelation of the CID spectra. The query spectrum is compared by using both mass information and fragmentation patterns. Fragmentation patterns are compared to each other using a correlation function. To evaluate the specificity of the approach, a set of 2180 tandem mass spectra obtained from both triple-quadrupole tandem mass spectrometers (TSQ) and quadrupole ion trap mass spectrometers (LCQ) was created. Comparisons are performed between tandem mass spectra obtained on the same instrument type as well as between different instrument types. Accurate and reliable comparisons are demonstrated in both types of analyses. The scores obtained in the cross-comparison of TSQ and LCQ tandem mass spectra of the same peptide are found to be slightly lower than comparisons performed with spectra obtained on the same instrument type. The method appears insensitive to variations in day-to-day performance of the instrument, minor variations in fragment ion abundance, and instrumental differences inherent in the same instrument model. The use of this method of comparison is demonstrated for library searching and subtractive analysis of tandem mass spectra obtained during LC/MS/MS experiments.     

Differentiation of lysine/glutamine in peptide sequence analysis by electrospray ionization sequential mass spectrometry coupled with a quadrupole ion trap

Electrospray ionization coupled to a quadrupole ion trap mass spectrometer is used to differentiate between the isobaric amino acids lysine and glutamine in sequence analysis of peptides. Collision-induced dissociation is used for fragmentation. Several isobaric peptides with one or more lysines or glutamines at different positions were investigated. The ambiguous amino acid either in the peptide chain or at the C- or N-terminus can be clearly identified based on specific side chain fragment ions resulting from MS3 or MS4 of B- and Y"-fragment ions.     

A MALDI probe for mass spectrometers

A new MALDI probe has been designed that uses transmission geometry. This geometry allows the probe to be fashioned after typical EI/CI solid probes which enables it to be introduced into spatially constrained ion source regions such as encountered in quadrupole ion trap mass spectrometers. In the probe design demonstrated here, light from a fiber optic irradiates the backside of a sample through a small piece of quartz on which the sample has been directly deposited. The performance characteristics exhibited by utilizing this probe for MALDI on a quadrupole ion trap mass spectrometer are similar to those which can be obtained through the traditional methods of implementing MALDI. Spectra have been obtained from 50 fmol of total loading of bombesin, MS/MS has been performed on 5 pmol of des-Arg9-bradykinin, and the analyte ion signal is shown to last for over 2500 laser shots for 2 pmol of bombesin. Optical micrographs showing the crystal distribution of a sample containing 2 pmol of bombesin have been obtained as a function of the number of laser shots for a single sample loading. Although this probe was designed for use with the quadrupole ion trap, it can be adapted for use with all types of mass spectrometers. Thus, with only one laser, one fiber optic, and this probe, MALDI can be performed on multiple instruments in a lab.     

Rapid identification of comigrating gel-isolated proteins by ion trap-mass spectrometry

In the search for novel nuclear binding proteins, two bands from a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel were analyzed and each was found to contain a number of proteins that subsequently were identified by tandem mass spectrometry (MS/MS) on a quadrupole ion trap instrument. The bands were digested with trypsin in situ on a polyvinylidene difluoride (PVDF) membrane following electroblot transfer. Analysis of a 2.5% aliquot of each peptide mixture by matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) followed by an initial database search with the peptide masses failed to identify the proteins. The peptides were separated by reversed-phase capillary high performance liquid chromatography (HPLC) in anticipation of subsequent Edman degradation, but mass analysis of the chromatographic fractions by MALDI-MS revealed multiple, coeluting peptides that precluded this approach. Selected fractions were analyzed by capillary HPLC-electrospray ionization-ion trap mass spectrometry. Tandem mass spectrometry provided significant fragmentation from which full or partial sequence was deduced for a number of peptides. Two stages of fragmentation (MS3) were used in one case to determine additional sequence. Database searches, each using a single peptide mass plus partial sequence, identified four proteins from a single electrophoretic band at 45 kDa, and four proteins from a second band at 60 kDa. Many of these proteins were derived from human keratin. The protein identifications were corroborated by the presence of multiple matching peptide masses in the MALDI-MS spectra. In addition, a novel sequence, not found in protein or DNA databases, was determined by interpretation of the MS/MS data. These results demonstrate the power of the quadrupole ion trap for the identification of multiple proteins in a mixture, and for de novo determination of peptide sequence. Reanalysis of the fragmentation data with a modified database searching algorithm showed that the same sets of proteins were identified from a limited number of fragment ion masses, in the absence of mass spectral interpretation or amino acid sequence. The implications for protein identification solely from fragment ion masses are discussed, including advantages for low signal levels, for a reduction of the necessary interpretation expertise, and for increased speed.     

De novo peptide sequencing in an ion trap mass spectrometer with 18O labeling

De novo peptide sequencing in an ion trap mass spectrometer coupled on-line with a capillary HPLC using 18O labeling provides a viable alternative to the method using the combination of nanospray, 18O labeling and a quadrupole/time-of-flight mass spectrometer. Seven to sixteen amino acid residues can be sequenced from the liquid chromatography/randem mass spectrometry (LC/MS/MS) spectra. This approach combines the benefit of capillary LC and the high sensitivity of the ion trap operated in the MS/MS mode. The wide availability of the LCQ mass spectrometer makes this approach readily adaptable to the biological mass spectrometry community.     

Collision-induced signal enhancement: a method to increase product ion intensities in MS/MS and MSn experiments

Collision-induced signal enhancement (CISE), a new technique to enhance the MSn capabilities of the quadrupole ion trap, is demonstrated. CISE is based on the chemistry, i.e., the dissociation pathways, of the analyte examined. Polysaccharides up to hexamers are used to demonstrate the capabilities of CISE to enhance signal in two distinct functional modes. Mode 1 CISE is designed to enhance the signal of an ion desired for MSn analysis. Mode 2 CISE is designed to enhance structurally significant product ions in an MS/MS spectrum. Two different approaches can be utilized to effect the two functional modes of CISE. Both approaches use conventional resonant excitation techniques to effect dissociation, which is performed nonanalytically, i.e., without isolation of the ions to be dissociated. The two approaches are (1) single-frequency resonance excitation, and (2) broad-band wave form resonant excitation. Experimental results for Mode 1 CISE analysis demonstrate up to a 17.3-fold signal increase for the single-frequency approach and 5.3-fold using broad-band excitation. Mode 2 CISE analysis shows up to a 16.3-fold increase in signal strength with single-frequency excitation and 3.3-fold using broad-band excitation.     

Fourier transform ion cyclotron resonance mass spectrometry: a primer

This review offers an introduction to the principles and generic applications of FT-ICR mass spectrometry, directed to readers with no prior experience with the technique. We are able to explain the fundamental FT-ICR phenomena from a simplified theoretical treatment of ion behavior in idealized magnetic and electric fields. The effects of trapping voltage, trap size and shape, and other nonidealities are manifested mainly as perturbations that preserve the idealized ion behavior modified by appropriate numerical correction factors. Topics include: effect of ion mass, charge, magnetic field, and trapping voltage on ion cyclotron frequency; excitation and detection of ICR signals; mass calibration; mass resolving power and mass accuracy; upper mass limit(s); dynamic range; detection limit, strategies for mass and energy selection for MSn; ion axialization, cooling, and remeasurement; and means for guiding externally formed ions into the ion trap. The relation of FT-ICR MS to other types of Fourier transform spectroscopy and to the Paul (quadrupole) ion trap is described. The article concludes with selected applications, an appendix listing accurate fundamental constants needed for ultrahigh-precision analysis, and an annotated list of selected reviews and primary source publications that describe in further detail various FT-ICR MS techniques and applications.     

Multipole storage assisted dissociation, a novel in-source dissociation technique for electrospray ionization generated ions

In this work we present a novel in-source dissociation scheme referred to as multipole storage assisted dissociation (MSAD) for electrospray ionization (ESI) generated ions in which dissociation is effected by employing extended ion accumulation intervals in a high pressure rf-only hexapole assembly prior to mass analysis. Following an extended ion accumulation interval in which ions are confined in the rf-only hexapole, ions are gated out of the hexapole, trapped, and mass analyzed in the trapped ion cell of a Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. The accumulation region is comprised of an rf-only hexapole ion guide which separates two electrodes, a biased skimmer cone, and an auxiliary 'gate' electrode at the low pressure end of the hexapole. This technique should be applicable to other mass spectrometry platforms compatible with pulsed ionization sources including quadrupole ion traps, and time-of-flight mass analyzers. This concept is demonstrated with the dissociation of a small protein in which selective fragmentation is observed at labile amino acid linkages producing primarily y-type fragment ions.     

Tandem mass spectrometry analysis of synthetic opioid peptide analogs

Five synthetic opioid peptides that were designed to have specific opioid receptor-binding properties were studied by low energy collision-induced dissociation (CID) tandem mass spectrometry (MS/MS). The MS/MS data are required for the analysis of those peptides in ovine plasma in a study to determine the placental transfer of the peptide to the fetus. The synthetic enkephalin-related peptides were: Tyr-D-Arg-Phe-Lys-NH2, (DALDA), N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH, (ICI 174,864), Tyr-D-Thr-Gly-Phe-Leu-Thr, (DTLET), Tyr-D-Pen-Gly-Phe-D-Pen-OH, (DPDPE), and D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2, (CTAP). Liquid secondary ion mass spectrometry (LSIMS) was used for sample desorption-ionization, and a hybrid (E1BE2qQ) tandem mass spectrometer was used to collect the product-ion spectra. A protonated molecule ion, [M + H]+, was observed for each peptide. Amino acid sequence-determining fragment ion were produced by CID and collected by MS/MS for the three linear peptides, and also for the two disulfide-bond-containing peptides in their unreduced and dithiothreitol (DTT)-reduced forms. The detection level for the [M + H]+ ion of DTLET was ca. 3 pmol; and the stabilities of the CTAP and ICI analogs in plasma were studied.     

A novel derivatization method with 5-bromonicotinic acid N-hydroxysuccinimide for determination of the amino acid sequences of peptides

We have developed a novel method that effectively identifies the N-terminal product ions produced in the tandem mass spectrometry (MS/MS) analysis of peptides done in conjunction with the specific derivatization of the N-terminal amino group using 5-bromonicotinic acid N-hydroxysuccinimide ester (BrNA-NHS). Electrospray ionization with low-energy collision-induced dissociation (CID) MS/MS clearly differentiated the N-terminal product ions labeled with the 5-bromonicotinyl group from other ions, on the basis of the appearance of CID peaks with a doublet pattern characteristically separated by 2 mass units produced by the equal natural abundances of 79Br and 81Br. The tracing of a series of these bromine-containing product ions allows the easy amino acid sequencing of peptides. Using Gln-Arg-Leu-Gln-Ser-Asn-Gln-Leu-Lys as the test peptide, we found that within 30 minutes at pH 6.5 and 37 degrees C its alpha-amino group was completely acylated with BrNA-NHS (peptide: BrNA-NHS = 1:40; mol/mol). The epsilon-amino group of the C-terminal lysine residue was less likely to be acylated under these conditions, being only partly modified (about 20%). This suggests the possibility of keeping the epsilon-amino group free from acylation. The method was successfully applied to the determination of the amino acid sequences of peptides from porcine kidney aminoacylase I produced by digestion with lysyl endopeptidase and with Staphylococus aureus V8 protease.     

Sodium-cationized oligosaccharides do not appear to undergo 'internal residue loss' rearrangement processes on tandem mass spectrometry

The phenomenon of 'internal residue loss' of protonated native- and per-O-methylated oligosaccharides has recently been described as occurring on high-energy collision conditions. Awareness of this phenomenon in the mass spectrometric analysis of oligosaccharides is of great importance since the rearrangement ions produced by this process may complicate monosaccharide sequence assignment. In this research, oligosaccharides having N-acetyl-glucosamine residues as the reducing or non-reducing terminal residue have been included in our MS/MS analyses in order to try to better understand the factors that influence 'internal residue loss'. Native and per-O-methylated compounds were submitted to positive and negative MS/MS, selecting protonated, sodium-cationized, or de-protonated pseudomolecular ions as precursors. High- and low-energy collision induced dissociation tandem mass spectrometry experiments were performed using a four sector instrument and a hybrid quadrupole time-of-flight mass spectrometer respectively. The phenomenon of 'internal residue loss' was not observed on either high- or low-energy CID-MS/MS when sodium-cationized precursor ions of either native or per-O-methylated oligosaccharides were examined. Similarly, MS/MS analysis performed in the negative ionization mode also failed to generate ions resulting from 'internal residue loss'. This combination of experiments therefore offers a way to be sure whether ions observed in the tandem mass spectra of protonated native or per-O-methylated oligosaccharides originate from 'internal residue loss' or from direct glycosidic linkage fragmentation.     

Confirmation of the structure of lipid A derived from the lipopolysaccharide of Rhodobacter sphaeroides by a combination of MALDI, LSIMS, and tandem mass spectrometry

The chemical structure of nontoxic diphosphoryl lipid A from Rhodobacter sphaeroides was confirmed using a combination of LSIMS (on a two-sector mass spectrometer) and MALDI (on time-of-flight and ion trap mass spectrometers) in conjunction with tandem mass spectrometry in both positive and negative ion modes. Accurate molecular weight measurement accompanied by the analysis of fragment ion masses yielded the composition of fatty acyl groups. Tandem experiments (collisionally induced dissociation of both quasimolecular and oxonium ions) were also performed, revealing the precise location and nature of the fatty acyl groups on the disaccharide backbone.     

Structural assignment of permethylated oligosaccharide subunits using sequential tandem mass spectrometry

The sequential tandem mass spectrometry (MSn) capabilities offered by quadrupole ion trap instruments have been explored in a systematic study of permethylated oligosaccharides. Under collision-induced dissociation, protonated molecular species generated in the electrospray ionization mode yield simple and predictable mass spectra. Information on sequence, branching, and, to some extent, interglycosidic linkages can be deduced from fragments resulting from the cleavage of glycosidic bonds. Simple rules for the structural assignment of carbohydrates have been established for the fragmentation of protonated species and subunits thereof and corroborated by 18O-labeling experiments. Moreover, sequential tandem mass spectrometry was demonstrated to allow the straightforward structural characterization of unknown carbohydrate moieties by comparing their CID spectra with those of a set of references. As the collision-induced dissociation patterns are not dependent on the number of prior tandem mass spectrometric steps, structures can be unambiguously assigned by match of the spectra. These findings establish the basis of MSn performed on a quadrupole ion trap instrument for elucidating structures of large carbohydrates, which can be virtually degraded in the mass spectrometer into smaller entities in one or several steps. This powerful technique has been applied, used in conjunction with specific CD3 labeling, to the characterization of series of subunits generated from fucosylated and sialylated oligosaccharides, which are among the most important structures as far as biological activities are concerned.     

Supercritical fluid extraction and negative ion electrospray liquid chromatography tandem mass spectrometry analysis of phenobarbital, butalbital, pentobarbital and thiopental in human serum

Four commonly used barbiturates (phenobarbital, butalbital, pentobarbital and thiopental) were analyzed in human serum using supercritical fluid extraction (SFE) and negative ionization LC/ESI-MS/MS. Barbital was used as the internal standard. Carbon dioxide SFE was performed at 40 degrees C and 500 atm, with a total extraction time of 35 min. The analytes were collected off-line in a liquid trap containing absolute methanol. Samples were then concentrated by vacuum centrifugation. The high performance liquid chromatography separation utilized gradient elution with a total analysis time of 21 min. The precursor and major product ions for the four barbiturates were monitored on a triple quadrupole mass spectrometer with negative ion electrospray ionization (ESI) in the multiple reaction monitoring mode as follows: (1) thiopental (m/z 241.20-->58.00), (2) phenobarbital (m/z 231.10-->188.0), (3) pentobarbital (m/z 225.10-->181.90) and (4) butalbital (m/z 222.80-->179.90). In the case of phenobarbital, pentobarbital and butalbital, the most abundant product ion arises from the loss of 43 u (HCNO loss). However, in the case of thiopental, the most abundant product ion was observed at m/z 58.0 (the [M-183]-ion, or NCS-). Mechanisms for the formation of the collision induced dissociation reaction products of these barbiturates are proposed.     

Class-selective collisionally activated dissociation/ion-molecule reactions of 4-quinolone antibiotics

A new combination of collisionally activated dissociation and ion-molecule reactions in a quadrupole ion trap mass spectrometer is developed for the class-selective determination of 4-quinolones, an important group of antibiotics. In this method, collisionally activated dissociation is used to promote dehydration of each protonated quinolone, and the resulting dehydrated fragment ion undergoes an addition reaction with an auxiliary reagent. The auxiliary reagents, including acetone, acetic anhydride, and n-propylamine, have structures that specifically allow formation of a cyclic adduct with the analyte ion. This sequential process is characteristic of a class of analyte molecules that have adjacent carboxylic acid and ketone functional groups anchored on a rigid ring and undergo facile, efficient dehydration.     

Exploring infrared wavelength matrix-assisted laser desorption/ionization of proteins with delayed-extraction time-of-flight mass spectrometry

We report a study of the application of delayed extraction (DE) to infrared-wavelength matrix-assisted time-of-flight mass spectrometry (IR-MALDI-TOF-MS) of proteins. The shapes of the spectral peaks obtained with DE-IR-MALDI-MS are compared with those obtained from the same samples and matrix using continuous extraction (CE) IR-MALDI-MS. Application of DE results in significant improvements in the peak resolution, revealing spectral features (in proteins with molecular masses < 12 kDa) that were not resolved in the corresponding CE-IR-Maldi mass spectra. Particularly significant is a series of peaks on the high mass side of the protonated protein peaks that arise through replacement of protons by adventitious sodium ions in the sample. We deduced that these sodium replacement species are a significant contributor to the broad tails (and resulting peak asymmetries) that are a feature of the DE-IR-MALDI mass spectra of proteins with molecular masses > or = 17 kDa. The peak width reduction observed in IR-MALDI by DE suggests that, as in UV-MALDI, the initial velocity distribution for ions produced in the MALDI process contributes to the peak broadness in the CE mass spectra. In a systematic comparison between DE UV-MALDI and DE IR-MALDI, we determined that photochemical matrix adduction is present in UV-MALDI but absent in IR-MALDI. In addition, we find that protein ions produced by IR irradiation are less internally excited (i.e., cooler), exhibiting less fragmentation, more Na+ replacement and/or unspecified noncovalent adduction, and more heme adduction with apomyoglobin. Thus, IR-MALDI appears to be a softer means for producing gas-phase protein ions than is UV-MALDI. It will be of considerable practical interest to determine whether large protein ions produced by IR-MALDI are sufficiently cool to survive transport through reflecting TOF mass spectrometers (without loss of small neutral species such as H2O, NH3, and CO2) and the extended time periods required for detection by quadrupole ion trap and Fourier transform ion cyclotron resonance mass analyzers.     

Instrumentation and applications of an automated C-terminal fragment peptide fractionator for C-terminal sequence analysis of proteins

A novel automated C-terminal fragment peptide fractionator has been constructed. Digests with lysyl endopeptidase were covalently immobilized on p-phenylene diisothiocyanate polymer beads. Only the C-terminal fragment, which contains no lysyl residue, was liberated by cleavage at the first peptide bond of the immobilized fragment peptides with trifluoroacetic acid, and it was automatically collected. The whole procedure was automatically and precisely performed under microprocessor control in a nitrogen atmosphere. The resulting fragment was sequenced without further purification. Sequences of both N- and C-terminal regions can be routinely obtained for ca. 100 pmol samples by the use of a conventional automated Edman-type protein sequencer.     

Protein identification by capillary zone electrophoresis/microelectrospray ionization-tandem mass spectrometry at the subfemtomole level

A method for the identification of proteins by their amino acid sequence at the low-femtomole to subfemtomole sensitivity level is described. It is based on an integrated system consisting of a capillary zone electrophoresis (CZE) instrument coupled to an electrospray ionization triple- quadrupole tandem mass spectrometer (ESI-MS/MS) via a microspray interface. The method consists of proteolytic fragmentation of a protein, peptide separation by CZE, analysis of separated peptides by ESI-MS/MS, and identification of the protein by correlation of the collision-induced dissociation (CID) patterns of selected peptides with the CID patterns predicted from all the isobaric peptides in a sequence database. Using standard peptides applied to a 20-microns-i.d. capillary, we demonstrate an ESI-MS limit of detection of less than 300 amol and CID spectra suitable for searching sequence databases obtained with 600 amol of sample applied to the capillary. Successful protein identification by the method was demonstrated by applying 50 and 38 fmol of a tryptic digest of the proteins beta-lactoglobulin and bovine serum albumin, respectively, to the system.     

Site-specific localization of Epstein-Barr virus in pharyngeal carcinomas

Electrospray ionization (ESI) Fourier transform ion cyclotron resonance (FTICR) mass spectrometry has been used to characterize heterotetrameric corynebacterial sarcosine oxidase. By using a conventional quadrupole mass spectrometer, no spectra for the intact complex could be obtained (i.e., electrospraying protein at neutral pH), but spectra showing the four protein subunits were obtained when electrospraying from acidic solution. Initial low resolution ESI-FTICR mass spectra of the intact heterotetramer revealed a typical narrow charge state distribution in the range 6000 < m/z < 9000, consistent with retention of a compact structure in the gas phase, and gave a mass measurement about 1000 u higher than predicted. Efficient in-trap clean up, based upon low energy collisionally induced dissociation of adducts, allowed significant improvement in mass measurement accuracy. The present results represent the largest heteromultimeric protein complex successfully analyzed using FTICR mass spectrometry, and clearly illustrate the importance of sample clean up methods for large molecule characterization.     

Electrospray ionization-ion trap mass spectrometry for structural analysis of complex N-linked glycoprotein oligosaccharides

Electrospray ionization-ion trap mass spectrometry, with its capacity to perform multiple stages of fragmentation (MSn), is demonstrated as an effective method for the structural characterization of permethylated N-linked complex glycoprotein oligosaccharides. Complex glycan structural features, such as N-acetyllactosamine antenane, neuraminic acids, and nonreducing terminal GlcNAc monosaccharides, commonly suppress cross-ring and core saccharide cleavages in traditional MS/MS experiments. Using ion trap mass spectrometry, removal of these substituents permits determination of branching patterns and intersaccharide linkages by MS3 and MS4. Both sequence and linkage data are obtained for N-acetyllactosamine and sialyl-N-acetyllactosamine oligosaccharide antennae from biantennary glycans using MS3, and the location of a bisecting GlcNAc residue is also established after exposing the core pentasaccharide. Higher-order experiments further illustrate the potential of electrospray ionization-quadrupole ion trap mass spectrometry for carbohydrate analysis, as MS8 is used to produce significant and otherwise unobtainable branching information for an oligosaccharide from chicken ovalbumin. These studies constitute further evidence of the unique role that ion trap mass spectrometry can assume in the area of oligosaccharide analysis.