Quantitative proteomics using 16O/18O labeling and linear ion trap mass spectrometry

Quantitative proteomics using stable isotopic 16O/18O labeling has emerged as a very powerful tool, since it has a number of advantages over other methods, including the simplicity of chemistry, the constant mass tag at the C termini and its general applicability. However, due to the small mass difference between labeled and unlabeled peptide species, this approach has usually been restricted to high-resolution mass spectrometers. In this study we explored whether the high-resolution scanning mode, together with the extremely high scanning speed of the linear IT allows the 16O/18O-labeling method to be used for accurate, large-scale quantitative analysis of proteomes. A protocol, including digestion, desalting, labeling, MS and quantitative analysis was developed and tested using protein standards and whole proteome extracts. Using this method we were able to identify and quantify 140 proteins from only 10 mug of a proteome extract from mesenchymal stem cells. Relative expression changes larger than twofold can be identified with this method at the 95% confidence level. Our results demonstrate that accurate quantitative analysis using 16O/18O labeling can be performed in the practice using linear IT MS, without compromising large-scale peptide identification efficiency.     

iTRAQ reagent-based quantitative proteomic analysis on a linear ion trap mass spectrometer

For proteomic analysis using tandem mass spectrometry, linear ion trap instruments provide unsurpassed sensitivity but unreliably detect low mass peptide fragments, precluding their use with iTRAQ reagent-labeled samples. Although the popular LTQ linear ion trap supports analyzing iTRAQ reagent-labeled peptides via pulsed Q dissociation, PQD, its effectiveness remains questionable. Using a standard mixture, we found careful tuning of relative collision energy necessary for fragmenting iTRAQ reagent-labeled peptides, and increasing microscan acquisition and repeat count improves quantification but identifies somewhat fewer peptides. We developed software to calculate abundance ratios via summing reporter ion intensities across spectra matching to each protein, thereby providing maximized accuracy. Testing found that results closely corresponded between analysis using optimized LTQ-PQD settings plus our software and using a Qstar instrument. Thus, we demonstrate the effectiveness of LTQ-PQD analyzing iTRAQ reagent-labeled peptides, and provide guidelines for successful quantitative proteomic studies.     

Gel-free analysis of the human brain proteome: application of liquid chromatography and mass spectrometry on biopsy and autopsy samples

This paper reports on the findings of the Biomedical Research Institute, as one of the participants in the pilot study of the HUPO Brain Proteome Project. A biopsy and autopsy study sample derived from human brain was distributed among the participants for proteomic analysis. In our laboratory, attention was focused on protein identification using the bottom-up shotgun approach. Protein extracts derived from both samples were trypsinized and analyzed separately by 2-D LC and MS. In a complementary approach, the tryptic digests were analyzed directly by LC-ESI-MS/MS and gas-phase fractionation in the mass spectrometer. Taken together, both proteomic approaches in combination with a stringent evaluation process, resulted in the confident identification of 209 proteins in the human brain samples under investigation.     

Comparison of alternative analytical techniques for the characterisation of the human serum proteome in HUPO Plasma Proteome Project

Based on the same HUPO reference specimen (C1-serum) with the six proteins of highest abundance depleted by immunoaffinity chromatography, we have compared five proteomics approaches, which were (1) intact protein fractionation by anion-exchange chromatography followed by 2-DE-MALDI-TOF-MS/MS for protein identification (2-DE strategy); (2) intact protein fractionation by 2-D HPLC followed by tryptic digestion of each fraction and microcapillary RP-HPLC/microESI-MS/MS identification (protein 2-D HPLC fractionation strategy); (3) protein digestion followed by automated online microcapillary 2-D HPLC (strong cation-exchange chromatography (SCX)-RPC) with IT microESI-MS/MS; (online shotgun strategy); (4) same as (3) with the SCX step performed offline (offline shotgun strategy) and (5) same as (4) with the SCX fractions reanalysed by optimised nanoRP-HPLC-nanoESI-MS/MS (offline shotgun-nanospray strategy). All five approaches yielded complementary sets of protein identifications. The total number of unique proteins identified by each of these five approaches was (1) 78, (2) 179, (3) 131, (4) 224 and (5) 330 respectively. In all, 560 unique proteins were identified. One hundred and sixty-five proteins were identified through two or more peptides, which could be considered a high-confidence identification. Only 37 proteins were identified by all five approaches. The 2-DE approach yielded more information on the pI-altered isoforms of some serum proteins and the relative abundance of identified proteins. The protein prefractionation strategy slightly improved the capacity to detect proteins of lower abundance. Optimising the separation at the peptide level and improving the detection sensitivity of ESI-MS/MS were more effective than fractionation of intact proteins in increasing the total number of proteins identified. Overall, electrophoresis and chromatography, coupled respectively with MALDI-TOF/TOF-MS and ESI-MS/MS, identified complementary sets of serum proteins.     

Analysis of iodinated peptides by LC-DAD/ESI ion trap mass spectrometry

The analysis of iodinated peptides resulting from chloramine-T (CAT), Iodo-Beads, Iodo-Gen and lactoperoxidase iodination reactions in the preparation of nanomole quantities 125I and 123I labelled tracers is described. Seven different model peptides were evaluated, varying in molecular weight from 294 (LY-dipeptide) to 2518 (obestatin containing 23 amino acid residues). Two different RP-C18 columns were used, each with a different gradient system based on aqueous formic acid and acetonitrile. Electrospray ionization (ESI) ion trap mass spectrometry was used for identification of the chromatographic eluting components of the reaction mixtures, while UV (DAD) served quantitative purposes. Non-, mono-, di-, tri- and tetra-iodinated peptides (respectively NIP, MIP, DIP, 3IP and 4IP) eluted in that order and were well separated from each other. An empirical model was derived. The applicability of this approach was demonstrated by the analysis of different reaction mixtures.     

Systematic comparison of a two-dimensional ion trap and a three-dimensional ion trap mass spectrometer in proteomics

The utility and advantages of the recently introduced two-dimensional quadrupole ion trap mass spectrometer in proteomics over the traditional three-dimensional ion trap mass spectrometer have not been systematically characterized. Here we rigorously compared the performance of these two platforms by using over 100,000 tandem mass spectra acquired with identical complex peptide mixtures and acquisition parameters. Specifically we compared four factors that are critical for a successful proteomic study: 1) the number of proteins identified, 2) sequence coverage or the number of peptides identified for every protein, 3) the data base matching SEQUEST X(corr) and S(p) score, and 4) the quality of the fragment ion series of peptides. We found a 4-6-fold increase in the number of peptides and proteins identified on the two-dimensional ion trap mass spectrometer as a direct result of improvement in all the other parameters examined. Interestingly more than 70% of the doubly and triply charged peptides, but not the singly charged peptides, showed better quality of fragmentation spectra on the two-dimensional ion trap. These results highlight specific advantages of the two-dimensional ion trap over the conventional three-dimensional ion traps for protein identification in proteomic experiments.     

The human erythrocyte proteome: analysis by ion trap mass spectrometry

This report describes an analysis of the red blood cell proteome by ion trap tandem mass spectrometry in line with liquid chromatography. Mature red blood cells lack all internal cell structures and consist of cytoplasm within a plasma membrane envelope. To maximize outcome, total red blood cell protein was divided into two fractions of membrane-associated proteins and cytoplasmic proteins. Both fractions were divided into subfractions, and proteins were identified in each fraction separately through tryptic digestion. Membrane protein digests were collected from externally exposed proteins, internally exposed proteins, "spectrin extract" mainly consisting of membrane skeleton proteins, and membrane proteins minus spectrin extract. Cytoplasmic proteins were divided into 21 fractions based on molecular mass by size exclusion chromatography. The tryptic peptides were separated by reverse-phase high-performance liquid chromatography and identified by ion trap tandem mass spectrometry. A total of 181 unique protein sequences were identified: 91 in the membrane fractions and 91 in the cytoplasmic fractions. Glyceraldehyde-3-phosphate dehydrogenase was identified with high sequence coverage in both membrane and cytoplasmic fractions. Identified proteins include membrane skeletal proteins, metabolic enzymes, transporters and channel proteins, adhesion proteins, hemoglobins, cellular defense proteins, proteins of the ubiquitin-proteasome system, G-proteins of the Ras family, kinases, chaperone proteins, proteases, translation initiation factors, and others. In addition to the known proteins, there were 43 proteins whose identification was not determined.     

A novel approach to denoising ion trap tandem mass spectra

Background  

Mass spectrometers can produce a large number of tandem mass spectra. They are unfortunately noise-contaminated. Noises can affect the quality of tandem mass spectra and thus increase the false positives and false negatives in the peptide identification. Therefore, it is appealing to develop an approach to denoising tandem mass spectra.     

Hybrid hierarchical clustering with applications to microarray data

In this paper, we propose a hybrid clustering method that combines the strengths of bottom-up hierarchical clustering with that of top-down clustering. The first method is good at identifying small clusters but not large ones; the strengths are reversed for the second method. The hybrid method is built on the new idea of a mutual cluster: a group of points closer to each other than to any other points. Theoretical connections between mutual clusters and bottom-up clustering methods are established, aiding in their interpretation and providing an algorithm for identification of mutual clusters. We illustrate the technique on simulated and real microarray datasets.     

Sequential abundant ion fragmentation analysis (SAIFA): an alternative approach for phosphopeptide identification using an ion trap mass spectrometer

Phosphorylation has been the most studied of all the posttranslational modifications of proteins. Mass spectrometry has emerged as a powerful tool for phosphomapping on proteins/peptides. Collision-induced dissociation (CID) of phosphopeptides leads to the loss of phosphoric or metaphosphoric acid as a neutral molecule, giving an intense neutral loss product ion in the mass spectrum. Dissociation of the neutral loss product ion identifies peptide sequence. This method of data-dependent constant neutral loss (DDNL) scanning analysis has been commonly used for mapping phosphopeptides. However, preferential losses of groups other than phosphate are frequently observed during CID of phosphopeptides. Ions that result from such losses are not identified during DDNL analysis due to predetermined scanning for phosphate loss. In this study, we describe an alternative approach for improved identification of phosphopeptides by sequential abundant ion fragmentation analysis (SAIFA). In this approach, there is no predetermined neutral loss molecule, thereby undergoing sequential fragmentation of abundant peak, irrespective of the moiety lost during CID. In addition to improved phosphomapping, the method increases the sequence coverage of the proteins identified, thereby increasing the confidence of protein identification. To the best of our knowledge, this is the first report to use SAIFA for phosphopeptide identification.     

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.     

Characterization of polypeptide antibiotics of the polymyxin series by liquid chromatography electrospray ionization ion trap tandem mass spectrometry.

A selective reversed phase liquid chromatography/mass spectrometry (LC/MSn) method is described for the identification of related compounds in commercial polymyxin B samples. Mass spectral data for these polypeptide antibiotics were acquired on a LCQ ion trap mass spectrometer equipped with an electrospray ionization probe operated in the positive ion mode. The LCQ ion trap is ideally suited for the identification of the related substances because it provides on-line LC/MSn capability. The main advantage of this hyphenated LC/MSn technique is the characterization of novel related substances without time-consuming isolation and purifications procedures. Using this method six novel related substances were partially identified in a polymyxin B bulk sample.     

Roles of dynamic linkage of stable attractors across cortical networks in recalling long-term memory

 We propose a neural network model for a category-association task. By simulating the model, neuronal relevance of cortical interactions to recalling long-term memory was investigated. The model consists of the left and right hemispheres, each of which has IT (inferotemporal cortex) and PC (prefrontal cortex) networks. Information about visual features and their categories were encoded into point attractors of the IT and PC networks, respectively. In the task, the IT network of the right hemisphere was stimulated with a cue feature. After a delay period, the IT network of the left hemisphere was simultaneously stimulated with the choice feature and an irrelevant feature. The cue and choice features belong to the same category, while the irrelevant feature belongs to another category. To complete the task, the IT network must select the point attractor corresponding to the choice feature. We demonstrate that the top-down pathway (PC-to-IT) triggers the retrieval of long-term memory of the choice feature from the IT, and the bottom-up pathway (IT-to-PC) contributes to the maintenance of the retrieved memory during the delay period. The key mechanism for the retrieval and maintenance of that memory is the dynamic linkage of attractors across separate cortical networks. We show that a single hemisphere is sufficient for the memory retrieval, but it is advantageous to use the two hemispheres because the retrieved memory is thereby retained with greater reliability until the brain chooses the choice feature. Received: 4 April 2001 / Accepted in revised form: 17 September 2002 / Published online: 20 January 2003 Correspondence to: O. Hoshino (e-mail: hoshino@cc.oita-u.ac.jp, Tel.: +81-97-554-7301, Fax: +81-97-554-7507)     

Two-dimensional components and hidden dependencies provide insight into ion channel gating mechanisms

Correlations between the durations of adjacent open and shut intervals recorded from ion channels contain information about the underlying gating mechanism. This study presents an additional approach to extracting the correlation information. Detailed correlation information is obtained directly from single-channel data and quantified in a manner that can provide insight into the connections among the states underlying the gating. The information is obtained independently of any specific kinetic scheme, except for the general assumption of Markov gating. The durations of adjacent open and shut intervals are binned into two-dimensional (2-D) dwell-time distributions. The 2-D (joint) distributions are fitted with sums of 2-D exponential components to determine the number of 2-D components, their volumes, and their open and closed time constants. The dependency of each 2-D component is calculated by comparing its observed volume to the volume that would be expected if open and shut intervals paired independently. The estimated component dependencies are then used to suggest gating mechanisms and to provide a powerful means of examining whether proposed gating mechanisms have the correct connections among states. The sensitivity of the 2-D method can identify hidden components and dependencies that can go undetected by previous correlation methods.     

100% protein sequence coverage: a modern form of surrealism in proteomics

This review intends not only to discuss the current possibilities to gain 100% sequence coverage for proteins, but also to point out the critical limits to such an attempt. The aim of 100% sequence coverage, as the review title already implies, seems to be rather surreal if the complexity and dynamic range of a proteome is taken into consideration. Nevertheless, established bottom-up shotgun approaches are able to roughly identify a complete proteome as exemplary shown by yeast. However, this proceeding ignores more or less the fact that a protein is present as various protein species. The unambiguous identification of protein species requires 100% sequence coverage. Furthermore, the separation of the proteome must be performed on the protein species and not on the peptide level. Therefore, top-down is a good strategy for protein species analysis. Classical 2D-electrophoresis followed by an enzymatic or chemical cleavage, which is a combination of top-down and bottom-up, is another interesting approach. Moreover, the review summarizes further top-down and bottom-up combinations and to which extent middle-down improves the identification of protein species. The attention is also focused on cleavage strategies other than trypsin, as 100% sequence coverage in bottom-up experiments is only obtainable with a combination of cleavage reagents.     

Sensitive detection of isoglobo and globo series tetraglycosylceramides in human thymus by ion trap mass spectrometry

Glycosphingolipids serve as ligands for receptors involved in signal transduction and immune recognition, as exemplified by isoglobotrihexosylceramide, an antigenic ligand for T cell receptors. Mechanistic studies on the regulation of isoglobotrihexosylceramide require biochemical measurement of its lysosomal precursor, isoglobotetraglycosylceramide. It remains a challenge to distinguish between complex tetraglycosylceramide glycosphingolipid isomers with the same sugar components but diverse internal linkages. Here we established a simple and sensitive method to separate globo- and isoglobotetraglycosylceramide by MS5 ion trap mass spectrometry, and report the identification of isoglobotetraglycosylceramide in a CHO cell line transfected by iGb3 synthase, as well as in human thymus.     

Characterization of protein glycosylation using chip-based infusion nanoelectrospray linear ion trap tandem mass spectrometry.

Mass spectrometry (MS) has the potential to revolutionize structural glycobiology and help in the understanding of how post-translation events such as glycosylation affect protein activities. Several approaches to determine the structure of glycopeptides have been used successfully including fast atom bombardment, matrix-assisted laser desorption ionization, and electrospray ionization with a wide variety of mass analyzers. However, the identification of glycopeptides in a complex mixture still remains a challenge. The source of this challenge is primarily due to the poor ionization efficiency and rapid degradation of glycopeptides. In this report we describe the use of a chip-based infusion nanoelectrospray ionization technique in combination with a recently developed linear ion trap for identification and characterization of glycosylation in complex mixtures. Two standard synthetic glycans were analyzed using multiple-stage fragmentation analysis in both positive and negative ionization modes. In addition, the high mannose type N-glycosylation in ribonuclease B (RNase B) was used to map the glycosylation site and obtain the glycan structures. We were able to map the glycosylation site and obtain the glycan structures in RNase B in a single analysis. The results reported here demonstrate that the fully automated chip-based nanoelectrospray linear ion trap platform is a valuable system for oligosaccharide analyses due to the unique MS/MS and MS(n) capability of the linear ion trap and the extended analysis time provided by the ionization technique.     

The selfie trap: A novel camera trap design for accurate small mammal identification

Camera traps are a popular tool for monitoring wildlife though they can fail to capture enough morphological detail for accurate small mammal species identification. Camera trapping small mammals is often limited by the inability of camera models to: (i) record at close distances; and (ii) provide standardised photos. This study aims to provide a camera trapping method that captures standardised images of the faces of small mammals for accurate species identification, with further potential for individual identification. A novel camera trap design coined the ‘selfie trap’ was developed. The selfie trap is a camera contained within an enclosed PVC pipe with a modified lens that produces standardised close images of small mammal species encountered in this study, including: Brown Antechinus (Antechinus stuartii), Bush Rat (Rattus fuscipes) and Sugar Glider (Petaurus breviceps). Individual identification was tested on the common arboreal Sugar Glider. Five individual Sugar Gliders were identified based on unique head stripe pelage. The selfie trap is an accurate camera trapping method for capturing detailed and standardised images of small mammal species. The design described may be useful for wildlife management as a reliable method for surveying small mammal species. However, intraspecies individual identification using the selfie trap requires further testing.     

Reliability of Quality Assessments in Research Synthesis: Securing the Highest Quality Bioinformation for HIT

Current trends in bio-medicine include research synthesis and dissemination of bioinformation by means of health (bio) information technology (H[b] IT). Research must secure the validity and reliability of assessment tools to quantify research quality in the pursuit of the best available evidence. Our concerted work in this domain led to the revision of three instruments for that purpose, including the stringent characterization of inter-rater reliability and coefficient of agreement. It is timely and critical to advance the methodological development of the science of research synthesis by strengthening the reliability of existing measure of research quality in order to ensure H[b] IT efficacy and effectiveness.     

Isolation and mass spectrometry characterization of molecular species of lactosylceramides using liquid chromatography-electrospray ion trap mass spectrometry

Reverse-phase liquid chromatography/electrospray ion trap mass spectrometry (LC-ESI-MSn) was established for identification of the molecular species of lactosylceramides. Lactosylceramides derived from porcine blood cells were separated on a CapcellPak C8 column using a mixture of methanol and 1 mM ammonium formate from the C16 to C26 fatty acyl chains based on the length of total carbon chains and the nature of sphingoid bases (w') and fatty acyl chains (Y0'-w') was identified by MS3 as their [M+H]+ ions. The same number of fatty acyl moieties appeared in the order of unsaturated, (2-)hydroxylated, and saturated components. The molecular species of lactosylceramides derived from porcine blood cells totaled more than 33 and included mainly C24:0-d18:1, Ch24:0-d18:1, Ch24:1-d18:1, C24:1-d18:1, and C22:0-d18:1 in addition to 28 minor species from C16:0 to C26:0 fatty acyl moieties. The molecular species of lactosylceramides in the membrane microdomain fraction of HL-60 cells (70% were differentiated into macrophage-lineage cells) were identified as C24:0-d18:1, C24:1-d18:1, C22:0-d18:1, C16:0-d18:1, and more than 21 other minor species. Our results suggest that reverse-phase LC-ESI-MSn is a useful and simple method for identification of lactosylceramide molecular species.