Conformational effect on the fragmentations of peptide derivatives in field desorption mass spectrometry

Fragmentations of N-benzyloxycarbonyl-protected tri-peptide ethyl esters containing proline at the P2 site were compared with those of the corresponding peptide derivatives containing no proline in field desorption mass spectrometry. The fragment ion [M-107]+ due to a loss of the benzyloxy group from a molecular ion was observed in the field desorption mass spectra for the peptides containing no proline, while it was not found in the peptides containing proline at all. These results suggest that the conformational difference of the peptide derivatives attributable to the existence of proline has an effect upon fragmentations in the field desorption ionizing process.     

Evaluation of different peptide fragmentation types and mass analyzers in data‐dependent methods using an Orbitrap Fusion Lumos Tribrid mass spectrometer

One of the major additions in MS technology has been the irruption of the Orbitrap mass analyzer, which has boosted the proteomics analyses of biological complex samples since its introduction. Here, we took advantage of the capabilities of the new Orbitrap Fusion Lumos Tribrid mass spectrometer to assess the performance of different data‐dependent acquisition methods for the identification and quantitation of peptides and phosphopeptides in single‐shot analysis of human whole cell lysates. Our study explored the capabilities of tri‐hibrid mass spectrometers for (phospho‐) peptide identification and quantitation using different gradient lengths, sample amounts, and combinations of different peptide fragmentation types and mass analyzers. Moreover, the acquisition of the same complex sample with different acquisition methods resulted in the generation of a dataset to be used as a reference for further analyses, and a starting point for future optimizations in particular applications.     

Low molecular mass peptide dendrimers that express antimicrobial properties

A series of low-generation dendrimeric peptides was synthesized in an attempt to evaluate their antimicrobial potency. All tested dendrimeric peptides in which lysine was a starting and branching element expressed moderate activity against Staphylococcus aureus NCTC 4163, and Escherichia coli NCTC 8196.     

18O-labeling quantitative proteomics using an ion trap mass spectrometer

We describe a method for simultaneous identification and quantitation of proteins within complex mixtures. The method consists of 18O-labeling, a simple stable isotope-coding that requires merely enzymatic digestion in 18O-water, in combination with a capillary-liquid chromatography electrospray ion-trap mass spectrometer. In a separate experiment using the same sample and a spike test, we demonstrate that the difference ration was calculated accurately using the 18O-labeling method even if the protein was part of a complex mixture. Our data also suggest that the accuracy of the quantitation can be improved by averaging the difference ratios of several peptides. In comparing our method with the isotope-coded affinity tag (ICAT) method, we show that the 18O-labeling method has the advantages of better recovery and fewer isotope effects. Therefore, the 18O-labeling method is a powerful tool for large-scale proteomics applications.     

Parts per million mass accuracy on an Orbitrap mass spectrometer via lock mass injection into a C-trap

Mass accuracy is a key parameter of mass spectrometric performance. TOF instruments can reach low parts per million, and FT-ICR instruments are capable of even greater accuracy provided ion numbers are well controlled. Here we demonstrate sub-ppm mass accuracy on a linear ion trap coupled via a radio frequency-only storage trap (C-trap) to the orbitrap mass spectrometer (LTQ Orbitrap). Prior to acquisition of a spectrum, a background ion originating from ambient air is first transferred to the C-trap. Ions forming the MS or MS(n) spectrum are then added to this species, and all ions are injected into the orbitrap for analysis. Real time recalibration on the "lock mass" by corrections of mass shift removes mass error associated with calibration of the mass scale. The remaining mass error is mainly due to imperfect peaks caused by weak signals and is addressed by averaging the mass measurement over the LC peak, weighted by signal intensity. For peptide database searches in proteomics, we introduce a variable mass tolerance and achieve average absolute mass deviations of 0.48 ppm (standard deviation 0.38 ppm) and maximal deviations of less than 2 ppm. For tandem mass spectra we demonstrate similarly high mass accuracy and discuss its impact on database searching. High and routine mass accuracy in a compact instrument will dramatically improve certainty of peptide and small molecule identification.     

Robust and sensitive iTRAQ quantification on an LTQ Orbitrap mass spectrometer

Isobaric stable isotope tagging reagents such as tandem mass tags or isobaric tags for relative and absolute quantification enable multiplexed quantification of peptides via reporter ion signals in the low mass range of tandem mass spectra. Until recently, the poor recovery of low mass fragments observed in tandem mass spectra acquired on ion trap mass spectrometers precluded the use of these reagents on this widely available instrument platform. The Pulsed Q Dissociation (PQD) technique allows negotiating this limitation but suffers from poor fragmentation efficiency, which has raised doubts in the community as to its practical utility. Here we show that by carefully optimizing instrument parameters such as collision energy, activation Q, delay time, ion isolation width, number of microscans, and number of trapped ions, low m/z fragment ion intensities can be generated that enable accurate peptide quantification at the 100 amol level. Side by side comparison of PQD on an LTQ Orbitrap with CID on a five-year old Q-Tof Ultima using complex protein digests shows that whereas precision of quantification of 10-15% can be achieved by both approaches, PQD quantifies twice as many proteins. PQD on an LTQ Orbitrap also outperforms "higher energy collision induced dissociation" on the same instrument using the recently introduced octapole collision cell in terms of lower limit of quantification. Finally, we demonstrate the significant analytical potential of iTRAQ quantification using PQD on an LTQ Orbitrap by quantitatively measuring the kinase interaction profile of the small molecule drug imatinib in K-562 cells. This article gives practical guidance for the implementation of PQD, discusses its merits, and for the first time, compares its performance to higher energy collision-induced dissociation.     

Gas phase hydrogen/deuterium exchange reactions of peptide ions in a quadrupole ion trap mass spectrometer

Hydrogen/deuterium exchange reactions of protonated and sodium cationized peptide molecules have been studied in the gas phase with a MALDI/quadrupole ion trap mass spectrometer. Unit-mass selected precursor ions were allowed to react with deuterated ammonia introduced into the trap cell by a pulsed valve. The reactant gas pressure, reaction time, and degree of the internal excitation of reactant ions were varied to explore the kinetics of the gas phase isotope exchange. Protonated peptide molecules exhibited a high degree of reactivity, some showing complete exchange of all labile hydrogen atoms. On the contrary, peptide molecules cationized with sodium exhibited only very limited reactivity, indicating a vast difference between the gas phase structures of the two ions. © 1997 Wiley-Liss Inc.     

Sequence determination by MALDI-TOF mass spectrometry of an insecticidal lentil peptide of the PA1b type

Mature seeds of lentil (Lens culinaris Medik.) were previously reported to contain an insecticidal cysteine-rich peptide, likely of the albumin-1 subunit b type. The purpose of this work was to determine the amino acid sequence of this insecticidal lentil peptide in an Eston lentil extract by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), after reduction of the disulfide bridges, alkylation of the cysteine residues and hydrolysis by pronase, trypsin, chymotrypsin and endoproteinase Asp-N. Sequences of key fragments were supported by monoisotopic mass measurements and by sequence ions from collision-induced dissociation (CID) experiments with a MALDI-TOF/TOF analyzer (MS/MS analysis). The new 37 amino acid sequence revealed strong similarities to a histidine-containing pea PA1b peptide and to soybean leginsulins but with a unique segment of RSSA in the middle. The lentil PA1b peptide sequence agreed completely with that derived from a L. culinaris genomic DNA sequence.     

Dual-source mass spectrometer with MALDI-LIT-ESI configuration

A novel linear ion trap (LIT) mass spectrometer with dual matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) ionization sources has been built in the MALDI-LIT-ESI configuration. The design features two independent ion source/ion optical channels connected to opposite ends of a single mass analyzer. The instrument consists of a commercial MALDI-LIT instrument modified by the addition of a home-built vacuum manifold, ion optical system, control electronics, and programming necessary to couple an atmospheric pressure interface to the commercial instrument. In addition to the added ESI functionality, the capabilities of the system also include simultaneous dual-channel ion introduction and analysis and high-duty cycle electronic switching (<1 s) between ion channels. Analytical and ion chemical applications of the dual-source system are explored. One analytical application is the enhanced protein sequence coverage achieved when using both ESI and MALDI to examine a tryptic digest of a six-protein mixture. The differences in the efficiency with which peptides in a mixture are ionized by the two methods give improved sequence coverage when both are applied. Other analytical applications include the use of the ions from one source as intensity or mass standards for the analyte ions from the other. An ion chemistry application involves the use of energy-resolved tandem mass spectrometry (MS/MS) to seek evidence for the generation of isomeric ions from a particular compound using the two ionization methods. A high level of agreement was achieved between the MS/MS spectra recorded under a variety of conditions after ESI and MALDI ionization; this provides evidence of the reproducibility and internal consistency of data from the dual source instrument. However, each of the peptides examined generated identical populations of structures in the two ionization methods under our conditions which are interpreted as involving slow cooling into the most stable minimum on the potential energy surface.     

A mass polarizability molecular beam spectrometer

Journal of Biological Physics - A molecular beam spectrometer using dielectrophoresis is described which should be useful in studies of molecular polarizabilities, dipole moments, rotational energy...     

Structural identification of the metabolites for strictosamide in rats bile by an ion trap-TOF mass spectrometer and mass defect filter technique

We report herein, a facile metabolite identification workflow on the antimicrobial strictosamide, which is derived from accurate mass measurement by a hybrid ion trap-TOF mass spectrometer. In step 1, the parent drug and metabolites in rat bile were separated on an HPLC column followed by ion trap-TOF mass spectrometer analysis after a single oral dose of 50mg/kg strictosamide. In step 2, mass defect filter technique, which enables high-resolution mass spectrometers to be utilized for detecting drug metabolites based on well-defined mass defect ranges, was used to find metabolites in the mass spectrum. In step 3, the differences of accurate masses and their mass fragmentation pattern among the parent drug and metabolites used to assign structures for the metabolites successfully. As a result, five metabolites of strictosamide were found in rat bile, and all the metabolites were reported for the first time.     

Discriminant models for high-throughput proteomics mass spectrometer data

We use several different multivariate analysis methods to discriminate between diseased and healthy patients using protein mass spectrometer data provided by Duke University. Two problems were presented by the university; one in which the responses (diseased or healthy) of the patients were not known and second, when the responses were known. In the latter case, the data can be used as a 'training' set. We attempted both problems. In particular, we use principle component analysis along with clustering methods to discriminate for the first problem set and partial least squares coupled with logistic and discriminant methods when the responses were known. In addition, we were able to detect regions of interest in the spectrum where there were differences in the protein patterns between healthy and diseased patients. There was considerable effort involved in the preprocessing of the data. We used a binning approach to reduce the number of variables rather than peak heights or peak areas. We performed a square root transformation on the data to help stabilize the variance; this in turn made a significant improvement in clustering results.     

Reproducibility of retention time using a splitless nanoLC coupled to an ESI-FTICR mass spectrometer.

Replicate injections of a myoglobin tryptic digest, ultrafiltrates of human serum, and ultrafiltrates of human plasma made on a splitless nanoscale liquid chromatography system coupled to a Fourier-transform ion cyclotron resonance mass spectrometer were utilized to assess analytical reproducibility. The mean (across 19 tryptic fragments detected in at least 3 of 24 replicate injections) of the 95% CIM of retention time is +/-6.3 sec and the maximum is +/-11.6 sec; when only those tryptic fragments that were found in 24 of 24 replicates are considered, the maximum 95% CIM of retention time drops to +/-6.7 sec. This represents a deviation of at most seven spectra. Similarly, in the serum (and plasma) filtrates, 95% of the 393 (312) species observed in 3 replicate injections had a 95% CIM of retention time of +/-22.0 (+/-18.5) sec or less. Ion abundance was similarly reproducible, with an average across those tryptic fragments observed in all 24 replicates of the coefficient of variation of ion abundance equal to 37.0%. This reproducibility represents a significant improvement over prior work, which required flow splitting in order to achieve nanoliter-per-minute flow rates. These improvements in retention time reproducibility will also be observed with mass spectrometers employing mass analyzers other than FT-ICR.     

Simulation of coherent energy transfer in an alpha-helical peptide by Fermi resonance.

A mechanism by which NH stretching quanta are coherently transported along a chain of hydrogen bonded peptide groups is demonstrated by classical simulation of a section of the alpha-helical peptide poly(L-alanine). Vibrational motion takes place on a complex energy surface constructed from earlier ab initio and empirical surfaces. A speculative hypothesis of the biological role of this mechanism is presented, and the critical parameters governing the dynamics are identified and discussed.     

Indirect estimation of cell mass and substrate concentration using a computer-coupled mass spectrometer

On-line estimation of cell mass and substrate concentration based on exhaust gas analysis was developed. The O₂, CO₂, H₂O, and N₂ contents at the inlet and outlet of fermentor, analyzed by a computer-coupled quadrupole mass spectrometer, were used to calculate the oxygen uptake rate and carbon dioxide evolution rate, and these rates were further used to evaluate cell mass and substrate concentration in a recombinant Escherichia coli fermentation. Cell mass, glucose concentration, specific growth rate, and specific consumption rate of glucose were well estimated by this method; the oxygen uptake rate gave more accurate estimates for these state variables than did the carbon dioxide evolution rate.     

New algorithm for 15N/14N quantitation with LC-ESI-MS using an LTQ-FT mass spectrometer

A new algorithm (QN) for the (15)N /(14)N quantitation of relative protein abundances in complex proteomic samples is described. QN takes advantage of the high resolution, mass accuracy and throughput of the hybrid mass spectrometer LTQ-FT MS. Peptide quantitation is based on MS peak intensity (measured in the FT MS), while peptide identification is performed in the MS/MS mode (measured in the LTQ linear ion trap). Accuracy of the protein abundance is enhanced by a novel scoring procedure, allowing filtering of less reliable measurements of peptide abundances. The performance of QN is illustrated in the relative quantitative analysis of M. acetivorans C2A cultures grown with carbon monoxide vs methanol as substrate. Roughly 1,000 proteins were quantitated with an average CV of 9% for the protein abundance ratios. QN performs quantitation without manual intervention, does not require high processing power, and generates files compatible with the Guidelines for Proteomic Data Publication.