共查询到20条相似文献,搜索用时 31 毫秒
1.
Michael A. Kuzyk Leanne B. Ohlund Monica H. Elliott Derek Smith Hong Qian Allen Delaney Christie L. Hunter Christoph H. Borchers 《Proteomics》2009,9(12):3328-3340
The peptide‐based quantitation accuracy and precision of LC‐ESI (QSTAR Elite) and LC‐MALDI (4800 MALDI TOF/TOF) were compared by analyzing identical Escherichia coli tryptic digests containing iTRAQ‐labeled peptides of defined abundances (1:1, 2.5:1, 5:1, and 10:1). Only 51.4% of QSTAR spectra were used for quantitation by ProteinPilot Software versus 66.7% of LC‐MALDI spectra. The average protein sequence coverages for LC‐ESI and LC‐MALDI were 24.0 and 18.2% (14.9 and 8.4 peptides per protein), respectively. The iTRAQ‐based expression ratios determined by ProteinPilot from the 57 467 ESI‐MS/MS and 26 085 MALDI‐MS/MS spectra were analyzed for measurement accuracy and reproducibility. When the relative abundances of peptides within a sample were increased from 1:1 to 10:1, the mean ratios calculated on both instruments differed by only 0.7–6.7% between platforms. In the 10:1 experiment, up to 64.7% of iTRAQ ratios from LC‐ESI MS/MS spectra failed S/N thresholds and were excluded from quantitation, while only 0.1% of the equivalent LC‐MALDI iTRAQ ratios were rejected. Re‐analysis of an archived LC‐MALDI sample set stored for 5 months generated 3715 MS/MS spectra for quantitation, compared with 3845 acquired originally, and the average ratios differed by only 3.1%. Overall, MS/MS‐based peptide quantitation performance of offline LC‐MALDI was comparable with on‐line LC‐ESI, which required threefold less time. However, offline LC‐MALDI allows the re‐analysis of archived HPLC‐separated samples. 相似文献
2.
Mark R. Condina Johan O. R. Gustafsson Manuela Klingler‐Hoffmann Christopher J. Bagley Shaun R. McColl Peter Hoffmann 《Proteomics》2010,10(13):2516-2530
The quality of MALDI‐TOF mass spectrometric analysis is highly dependent on the matrix and its deposition strategy. Although different matrix‐deposition methods have specific advantages, one major problem in the field of proteomics, particularly with respect to quantitation, is reproducibility between users or laboratories. Compounding this is the varying crystal homogeneity of matrices depending on the deposition strategy used. Here, we describe a novel optimised matrix‐deposition strategy for LC‐MALDI‐TOF/TOF MS using an automated instrument that produces a nebulised matrix “mist” under controlled atmospheric conditions. Comparisons of this with previously reported strategies showed the method to be advantageous for the atypical matrix, 2,5‐DHB, and improved phosphopeptide ionisation when compared with deposition strategies for CHCA. This optimised DHB matrix‐deposition strategy with LC‐MALDI‐TOF/TOF MS, termed EZYprep LC, was subsequently optimised for phosphoproteome analysis and compared to LC‐ESI‐IT‐MS and a previously reported approach for phosphotyrosine identification and characterisation. These methods were used to map phosphorylation on epidermal growth factor‐stimulated epidermal growth factor receptor to gauge the sensitivity of the proposed method. EZYprep DHB LC‐MALDI‐TOF/TOF MS was able to identify more phosphopeptides and characterise more phosphorylation sites than the other two proteomic strategies, thus proving to be a sensitive approach for phosphoproteome analysis. 相似文献
3.
Phosphorylation is a reversible posttranslational protein modification which plays a pivotal role in intracellular signaling. Despite extensive efforts, phosphorylation site mapping of proteomes is still incomplete motivating the exploration of alternative methods that complement existing workflows. In this study, we compared tandem mass spectrometry (MS/MS) on matrix assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) and nano‐electrospray ionization (nESI) Orbitrap instruments with respect to their ability to identify phosphopeptides from complex proteome digests. Phosphopeptides were enriched from tryptic digests of cell lines using Fe‐IMAC column chromatography and subjected to LC‐MS/MS analysis. We found that the two analytical workflows exhibited considerable orthogonality. For instance, MALDI‐TOF MS/MS favored the identification of phosphopeptides encompassing clear motif signatures for acidic residue directed kinases. The extent of orthogonality of the two LC‐MS/MS systems was comparable to that of using alternative proteases such as Asp‐N, Arg‐C, chymotrypsin, Glu‐C and Lys‐C on just one LC‐MS/MS instrument. Notably, MALDI‐TOF MS/MS identified an unexpectedly high number and percentage of phosphotyrosine sites (~20% of all sites), possibly as a direct consequence of more efficient ionization. The data clearly show that LC‐MALDI MS/MS can be a useful complement to LC‐nESI MS/MS for phosphoproteome mapping and particularly so for acidic and phosphotyrosine containing peptides. 相似文献
4.
Javed Iqbal Wang Li Kaleem Ullah Murtaza Hasan Guo Linna Umer Awan Yongqian Zhang Sajida Batool Hong Qing Yulin Deng 《Proteomics》2013,13(16):2455-2468
The proteomic profile of hypothalamus, a key organ of CNS, is explored here by employing two widely used MS techniques, i.e. HPLC/ESI‐ion trap and HPLC/ESI‐quadrupole‐TOF MS. Strong cation exchange is used for the fractionation of peptides and protein search engine MASCOT is employed for data query. One hundred and thirty six proteins with 10 973 peptides were identified by HPLC/ESI‐ion trap MS, while 140 proteins with 32 183 peptides were characterized by HPLC/ESI‐quadrupole‐TOF MS. Among the total 198 proteins identified in both experiments, 78 proteins were common in both sets of conditions. The rest of the 120 proteins were identified distinctly in both MS strategies, i.e. 58 unique proteins were found using the quadrupole‐TOF while 62 were found with the HPLC/ESI‐ion trap. Moreover, these proteins were classified into groups based on their functions performed in the body. Results presented here identified some important signal and cellular defense proteins inevitable for survival in stressed conditions. Additionally, it is also shown that any single MS strategy is not reliable for good results due to loss of data depending on sensitivity of the instrument used. 相似文献
5.
Pan S Rush J Peskind ER Galasko D Chung K Quinn J Jankovic J Leverenz JB Zabetian C Pan C Wang Y Oh JH Gao J Zhang J Montine T Zhang J 《Journal of proteome research》2008,7(2):720-730
Targeted quantitative proteomics by mass spectrometry aims to selectively detect one or a panel of peptides/proteins in a complex sample and is particularly appealing for novel biomarker verification/validation because it does not require specific antibodies. Here, we demonstrated the application of targeted quantitative proteomics in searching, identifying, and quantifying selected peptides in human cerebrospinal spinal fluid (CSF) using a matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometer (MALDI TOF/TOF)-based platform. The approach involved two major components: the use of isotopic-labeled synthetic peptides as references for targeted identification and quantification and a highly selective mass spectrometric analysis based on the unique characteristics of the MALDI instrument. The platform provides high confidence for targeted peptide detection in a complex system and can potentially be developed into a high-throughput system. Using the liquid chromatography (LC) MALDI TOF/TOF platform and the complementary identification strategy, we were able to selectively identify and quantify a panel of targeted peptides in the whole proteome of CSF without prior depletion of abundant proteins. The effectiveness and robustness of the approach associated with different sample complexity, sample preparation strategies, as well as mass spectrometric quantification were evaluated. Other issues related to chromatography separation and the feasibility for high-throughput analysis were also discussed. Finally, we applied targeted quantitative proteomics to analyze a subset of previously identified candidate markers in CSF samples of patients with Parkinson's disease (PD) at different stages and Alzheimer's disease (AD) along with normal controls. 相似文献
6.
A notable inefficiency of shotgun proteomics experiments is the repeated rediscovery of the same identifiable peptides by sequence database searching methods, which often are time-consuming and error-prone. A more precise and efficient method, in which previously observed and identified peptide MS/MS spectra are catalogued and condensed into searchable spectral libraries to allow new identifications by spectral matching, is seen as a promising alternative. To that end, an open-source, functionally complete, high-throughput and readily extensible MS/MS spectral searching tool, SpectraST, was developed. A high-quality spectral library was constructed by combining the high-confidence identifications of millions of spectra taken from various data repositories and searched using four sequence search engines. The resulting library consists of over 30,000 spectra for Saccharomyces cerevisiae. Using this library, SpectraST vastly outperforms the sequence search engine SEQUEST in terms of speed and the ability to discriminate good and bad hits. A unique advantage of SpectraST is its full integration into the popular Trans Proteomic Pipeline suite of software, which facilitates user adoption and provides important functionalities such as peptide and protein probability assignment, quantification, and data visualization. This method of spectral library searching is especially suited for targeted proteomics applications, offering superior performance to traditional sequence searching. 相似文献
7.
生物质谱技术是蛋白质组学的支撑技术.详细论述了质谱技术的分类与基本分析原理,重点论述了质谱技术的发展变化,包括基质辅助激光解吸飞行时间质谱技术,电喷雾质谱技术,MALDI-Q-TOF和MAL-DI-TOF-TOF等质谱技术,以及质谱技术在蛋白质组学研究中的应用与未来的发展和挑战. 相似文献
8.
Modern nano‐HPLC systems are capable of extremely precise control of solvent gradients, allowing high‐resolution separation of peptides. Most proteomics laboratories use a simple linear analytical gradient for nano‐LC‐MS/MS experiments, though recent evidence indicates that optimized non‐linear gradients result in increased peptide and protein identifications from cell lysates. In concurrent work, we examined non‐linear gradients for the analysis of samples fractionated at the peptide level, where the distribution of peptide retention times often varies by fraction. We hypothesized that greater coverage of these samples could be achieved using per‐fraction optimized gradients. We demonstrate that the optimized gradients improve the distribution of peptides throughout the analysis. Using previous generation MS instrumentation, a considerable gain in peptide and protein identifications can be realized. With current MS platforms that have faster electronics and achieve shorter duty cycle, the improvement in identifications is smaller. Our gradient optimization method has been implemented in a simple graphical tool (GOAT) that is MS‐vendor independent, does not require peptide ID input, and is freely available for non‐commercial use at http://proteomics.swmed.edu/goat/ 相似文献
9.
We applied the improved sensitivity and soft ionization characteristics of electrospray Ionization (ESI)-MS/MS and matrix-assisted laser desorption/ionization(MALDI)-time of flight (TOF) mass spectrometry (MS) to analysis of the GPI-anchored C-terminal peptide derived from 5'-nucleotidase. ESI-MS/MS analysis was applied to the core structure (MW, 2,743). In the collision-induced dissociation (CID) spectrum, single-charged ions such as m/z 162 (glucosamine), 286 (mannose-phosphate-ethanolamine), and 447 ([mannose-phosphate-ethanolamine]-glucosamine) were clearly detected as characteristic fragment ions of the GPI-anchored peptide. On MALDI-TOF-MS analysis, heterogeneous peaks of GPI-anchored peptides were detected as single-charged ions in the positive mode. Product ions were obtained by post-source decay (PSD) of m/z 2,905 using curved field reflectron of TOF-MS. Most of the expected product ions derived from the GPI-anchored peptide, containing the core structure and an additional mannose side chain, were successively obtained. Thus, ESI-MS/MS and MALDI-TOF-PSD-MS proved to be effective and sensitive methods for analyzing the GPI-anchored peptide structure with less than 10 pmol of sample. These characteristic fragments or fragmentation patterns seem to be very useful for identification of GPI-anchored C-terminal peptides derived from any kind of GPI-anchored protein. 相似文献
10.
Amaya Albalat Angelique Stalmach Vasiliki Bitsika Justyna Siwy Joost P. Schanstra Alexandros D. Petropoulos Antonia Vlahou Joachim Jankowski Frederik Persson Peter Rossing Thorsten W. Jaskolla Harald Mischak Holger Husi 《Proteomics》2013,13(20):2967-2975
Proteomic profiling by MALDI‐TOF MS presents various advantages (speed of analysis, ease of use, relatively low cost, sensitivity, tolerance against detergents and contaminants, and possibility of automation) and is being currently used in many applications (e.g. peptide/protein identification and quantification, biomarker discovery, and imaging MS). Earlier studies by many groups indicated that moderate reproducibility in relative peptide quantification is a major limitation of MALDI‐TOF MS. In the present work, we examined and demonstrate a clear effect, in cases apparently random, of sample dilution in complex samples (urine) on the relative quantification of peptides by MALDI‐TOF MS. Results indicate that in urine relative abundance of peptides cannot be assessed with confidence based on a single MALDI‐TOF MS spectrum. To account for this issue, we developed and propose a novel method of determining the relative abundance of peptides, taking into account that peptides have individual linear quantification ranges in relation to sample dilution. We developed an algorithm that calculates the range of dilutions at which each peptide responds in a linear manner and normalizes the received peptide intensity values accordingly. This concept was successfully applied to a set of urine samples from patients diagnosed with diabetes presenting normoalbuminuria (controls) and macroalbuminuria (cases). 相似文献
11.
Over 4100 protein identifications from a Xenopus laevis fertilized egg digest using reversed‐phase chromatographic prefractionation followed by capillary zone electrophoresis–electrospray ionization–tandem mass spectrometry analysis
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Xiaojing Yan Liangliang Sun Guijie Zhu Olivia F. Cox Norman J. Dovichi 《Proteomics》2016,16(23):2945-2952
A tryptic digest generated from Xenopus laevis fertilized embryos was fractionated by RPLC. One set of 30 fractions was analyzed by 100‐min CZE‐ESI‐MS/MS separations (50 h total instrument time), and a second set of 15 fractions was analyzed by 3‐h UPLC‐ESI‐MS/MS separations (45 h total instrument time). CZE‐MS/MS produced 70% as many protein IDs (4134 versus 5787) and 60% as many peptide IDs (22 535 versus 36 848) as UPLC‐MS/MS with similar instrument time (50 h versus 45 h) but with 50 times smaller total consumed sample amount (1.5 μg versus 75 μg). Surprisingly, CZE generated peaks that were 25% more intense than UPLC for peptides that were identified by both techniques, despite the 50‐fold lower loading amount; this high sensitivity reflects the efficient ionization produced by the electrokinetically pumped nanospray interface used in CZE. This report is the first comparison of CZE‐MS/MS and UPLC‐MS/MS for large‐scale eukaryotic proteomic analysis. The numbers of protein and peptide identifications produced by CZE‐ESI‐MS/MS approach those produced by UPLC‐MS/MS, but with nearly two orders of magnitude lower sample amounts. 相似文献
12.
Stefan K. Maier Ksenia Bashkueva Christoph Rösli Arne Skerra Bernhard Kuster 《Proteomics》2014,14(21-22):2427-2431
Mass spectrometers equipped with matrix‐assisted laser desorption/ionization (MALDI‐MS) require frequent multipoint calibration to obtain good mass accuracy over a wide mass range and across large numbers of samples. In this study, we introduce a new synthetic peptide mass calibration standard termed PAS‐cal tailored for MALDI‐MS based bottom‐up proteomics. This standard consists of 30 peptides between 8 and 37 amino acids long and each constructed to contain repetitive sequences of Pro, Ala and Ser as well as one C‐terminal arginine residue. MALDI spectra thus cover a mass range between 750 and 3200 m/z in MS mode and between 100 and 3200 m/z in MS/MS mode. Our results show that multipoint calibration of MS spectra using PAS‐cal peptides compares well to current commercial reagents for protein identification by PMF. Calibration of tandem mass spectra from LC‐MALDI experiments using the longest peptide, PAS‐cal37, resulted in smaller fragment ion mass errors, more matching fragment ions and more protein and peptide identifications compared to commercial standards, making the PAS‐cal standard generically useful for bottom‐up proteomics. 相似文献
13.
Tryptic digestion of proteins continues to be a workhorse of proteomics. Traditional tryptic digestion requires several hours to generate an adequate protein digest. A number of enhanced accelerated digestion protocols have been developed in recent years. Nonetheless, a need still exists for new digestion strategies that meet the demands of proteomics for high-throughput and rapid detection and identification of proteins. We performed an evaluation of direct tryptic digestion of proteins on a MALDI target plate and the potential for integrating RP HPLC separation of protein with on-target tryptic digestion in order to achieve a rapid and effective identification of proteins in complex biological samples. To this end, we used a Tempo HPLC/MALDI target plate deposition hybrid instrument (ABI). The technique was evaluated using a number of soluble and membrane proteins and an MRC5 cell lysate. We demonstrated that direct deposition of proteins on a MALDI target plate after reverse-phase HPLC separation and subsequent tryptic digestion of the proteins on the target followed by MALDI TOF/TOF analysis provided substantial data (intact protein mass, peptide mass and peptide fragment mass) that allowed a rapid and unambiguous identification of proteins. The rapid protein separation and direct deposition of fractions on a MALDI target plate provided by the RP HPLC combined with off-line interfacing with the MALDI MS is a unique platform for rapid protein identification with improved sequence coverage. This simple and robust approach significantly reduces the sample handling and potential loss in large-scale proteomics experiments. This approach allows combination of peptide mass fingerprinting (PMF), MS/MS peptide fragment fingerprinting (PPF) and whole protein MS for both protein identification and structural analysis of proteins. 相似文献
14.
Ali Tiss Celia Smith Usha Menon Ian Jacobs John F. Timms Rainer Cramer 《Proteomics》2010,10(18):3388-3392
MALDI MS profiling, using easily available body fluids such as blood serum, has attracted considerable interest for its potential in clinical applications. Despite the numerous reports on MALDI MS profiling of human serum, there is only scarce information on the identity of the species making up these profiles, particularly in the mass range of larger peptides. Here, we provide a list of more than 90 entries of MALDI MS profile peak identities up to 10 kDa obtained from human blood serum. Various modifications such as phosphorylation were detected among the peptide identifications. The overlap with the few other MALDI MS peak lists published so far was found to be limited and hence our list significantly extends the number of identified peaks commonly found in MALDI MS profiling of human blood serum. 相似文献
15.
Balazy M 《Prostaglandins & other lipid mediators》2004,73(3-4):173-180
Recent advancements in mass spectrometry, especially the development of electrospray tandem mass spectrometry (ESI/LC/MS2) and matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI/TOF), have greatly facilitated analysis of complex biomolecules. It has now become possible to profile, in relatively short periods of time, large multicomponent groups of compounds biosynthesized by biological systems. The efficiency and accuracy of analysis have led to the development of new concepts of mass spectrometric profiling, mapping, and imaging. Profiling of proteins in biological material (proteomics) has become a widely accepted strategy for identification of mechanisms involved in the biochemistry of disease processes, and has become a novel tool for unraveling new drug targets. Evolution of proteomics has relied on ESI/LC/MS2 and MALDI/TOF, techniques that are also useful in the novel area of quantitative proteomics. 相似文献
16.
Shunsuke Aburaya Wataru Aoki Hiroyoshi Minakuchi 《Bioscience, biotechnology, and biochemistry》2017,81(12):2237-2243
In proteomics, more than 100,000 peptides are generated from the digestion of human cell lysates. Proteome samples have a broad dynamic range in protein abundance; therefore, it is critical to optimize various parameters of LC–ESI–MS/MS to comprehensively identify these peptides. However, there are many parameters for LC–ESI–MS/MS analysis. In this study, we applied definitive screening design to simultaneously optimize 14 parameters in the operation of monolithic capillary LC–ESI–MS/MS to increase the number of identified proteins and/or the average peak area of MS1. The simultaneous optimization enabled the determination of two-factor interactions between LC and MS. Finally, we found two parameter sets of monolithic capillary LC–ESI–MS/MS that increased the number of identified proteins by 8.1% or the average peak area of MS1 by 67%. The definitive screening design would be highly useful for high-throughput analysis of the best parameter set in LC–ESI–MS/MS systems. 相似文献
17.
Protein labeling by iTRAQ: a new tool for quantitative mass spectrometry in proteome research 总被引:3,自引:0,他引:3
A novel, MS-based approach for the relative quantification of proteins, relying on the derivatization of primary amino groups in intact proteins using isobaric tag for relative and absolute quantitation (iTRAQ) is presented. Due to the isobaric mass design of the iTRAQ reagents, differentially labeled proteins do not differ in mass; accordingly, their corresponding proteolytic peptides appear as single peaks in MS scans. Because quantitative information is provided by isotope-encoded reporter ions that can only be observed in MS/MS spectra, we analyzed the fragmentation behavior of ESI and MALDI ions of peptides generated from iTRAQ-labeled proteins using a TOF/TOF and/or a QTOF instrument. We observed efficient liberation of reporter ions for singly protonated peptides at low-energy collision conditions. In contrast, increased collision energies were required to liberate the iTRAQ label from lysine side chains of doubly charged peptides and, thus, to observe reporter ions suitable for relative quantification of proteins with high accuracy. We then developed a quantitative strategy that comprises labeling of intact proteins by iTRAQ followed by gel electrophoresis and peptide MS/MS analyses. As proof of principle, mixtures of five different proteins in various concentration ratios were quantified, demonstrating the general applicability of the approach presented here to quantitative MS-based proteomics. 相似文献
18.
Hofmann S Glückmann M Kausche S Schmidt A Corvey C Lichtenfels R Huber C Albrecht C Karas M Herr W 《Molecular & cellular proteomics : MCP》2005,4(12):1888-1897
Identification of major histocompatibility complex (MHC)-associated peptides recognized by T-lymphocytes is a crucial prerequisite for the detection and manipulation of specific immune responses in cancer, viral infections, and autoimmune diseases. Unfortunately immunogenic peptides are less abundant species present in highly complex mixtures of MHC-extracted material. Most peptide identification strategies use microcapillary LC coupled to nano-ESI MS/MS in a challenging on-line approach. Alternatively MALDI PSD analysis has been applied for this purpose. We report here on the first off-line combination of nanoscale (nano) LC and MALDI TOF/TOF MS/MS for the identification of naturally processed MHC peptide ligands. These peptides were acid-eluted from human leukocyte antigen (HLA)-A2, HLA-A3, and HLA-B/-C complexes separately isolated from a renal cell carcinoma cell lysate using HLA allele-specific antibodies. After reversed-phase HPLC, peptides were further fractionated via nano-LC. This additional separation step provided a substantial increase in the number of detectable candidate species within the complex peptide pools. MALDI MS/MS analysis on nano-LC-separated material was then sufficiently sensitive to rapidly identify more than 30 novel HLA-presented peptide ligands. Peptide sequences contained perfect anchor amino acid residues described previously for HLA-A2, HLA-A3, and HLA-B7. The most promising candidate for a T-cell epitope is an HLA-B7-binding nonamer peptide derived from the tumor-associated gene NY-BR-16. To demonstrate the sensitivity of our approach we characterized peptides binding to HLA-C molecules that are usually expressed at the cell surface at approximately only 10% the levels of HLA-A or HLA-B. In fact, multiple renal cell carcinoma peptides were identified that contained anchor amino acid residues of HLA-Cw5 and HLA-Cw7. We conclude that the nano-LC MALDI MS/MS approach is a sensitive tool for the rapid and automated identification of MHC-associated tumor peptides. 相似文献
19.
Liquid chromatography MALDI MS/MS for membrane proteome analysis 总被引:3,自引:0,他引:3
Membrane proteins play critical roles in many biological functions and are often the molecular targets for drug discovery. However, their analysis presents a special challenge largely due to their highly hydrophobic nature. We present a surfactant-aided shotgun proteomics approach for membrane proteome analysis. In this approach, membrane proteins were solubilized and digested in the presence of SDS followed by newly developed auto-offline liquid chromatography/matrix-assisted laser desorption ionization (LC/MALDI) tandem MS analysis. Because of high tolerance of MALDI to SDS, one-dimensional (1D) LC separation can be combined with MALDI for direct analysis of protein digests containing SDS, without the need for extensive sample cleanup. In addition, the heated droplet interface used in LC/MALDI can work with high flow LC separations, allowing a relatively large amount of protein digest to be used for 1D LC/MALDI which facilitates the detection of low abundance proteins. The proteome identification results obtained by LC/MALDI are compared to the gel electrophoresis/MS method as well as the shotgun proteomics method using 2D LC/electrospray ionization MS. It is demonstrated that, while LC/MALDI provides more extensive proteome coverage compared to the other two methods, these three methods are complementary to each other and a combination of these methods should provide a more comprehensive membrane proteome analysis. 相似文献
20.
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. 相似文献