Current methodologies for protein quantitation include 2-dimensional gel electrophoresis techniques, metabolic labeling, and stable isotope labeling methods to name only a few. The current literature illustrates both pros and cons for each of the previously mentioned methodologies. Keeping with the teachings of William of Ockham, "with all things being equal the simplest solution tends to be correct", a simple LC/MS based methodology is presented that allows relative changes in abundance of proteins in highly complex mixtures to be determined. Utilizing a reproducible chromatographic separations system along with the high mass resolution and mass accuracy of an orthogonal time-of-flight mass spectrometer, the quantitative comparison of tens of thousands of ions emanating from identically prepared control and experimental samples can be made. Using this configuration, we can determine the change in relative abundance of a small number of ions between the two conditions solely by accurate mass and retention time. Employing standard operating procedures for both sample preparation and ESI-mass spectrometry, one typically obtains under 5 ppm mass precision and quantitative variations between 10 and 15%. The principal focus of this paper will demonstrate the quantitative aspects of the methodology and continue with a discussion of the associated, complementary qualitative capabilities.
The detection, correlation, and comparison of peptide and product ions from a data independent LC-MS acquisition strategy with data dependent LC-MS/MS is described. The data independent mode of acquisition differs from an LC-MS/MS data acquisition since no ion transmission window is applied with the first mass analyzer prior to collision induced disassociation. Alternating the energy applied to the collision cell, between low and elevated energy, on a scan-to-scan basis, provides accurate mass precursor and associated product ion spectra from every ion above the LOD of the mass spectrometer. The method therefore provides a near 100% duty cycle, with an inherent increase in signal intensity due to the fact that both precursor and product ion data are collected on all isotopes of every charge-state across the entire chromatographic peak width. The correlation of product to precursor ions, after deconvolution, is achieved by using reconstructed retention time apices and chromatographic peak shapes. Presented are the results from the comparison of a simple four protein mixture, in the presence and absence of an enzymatically digested protein extract from Escherichia coli. The samples were run in triplicate by both data dependant analysis (DDA) LC-MS/MS and data-independent, alternate scanning LC-MS. The detection and identification of precursor and product ions from the combined DDA search results of the four protein mixture were used for comparison to all other data. Each individual set of data-independent LC-MS data provides a more comprehensive set of detected ions than the combined peptide identifications from the DDA LC-MS/MS experiments. In the presence of the complex E. coli background, over 90% of the monoisotopic masses from the combined LC-MS/MS identifications were detected at the appropriate retention time. Moreover, the fragmentation pattern and number of associated elevated energy product ions in each replicate experiment was found to be very similar to the DDA identifications. In the case of the corresponding individual DDA LC-MS/MS experiment, 43% of the possible detectable peptides of interest were identified. The presented data illustrates that the time-aligned data from data-independent alternate scanning LC-MS experiments is highly comparable to the data obtained via DDA. The obtained information can therefore be effectively and correctly deconvolved to correlate product ions with parent precursor ions. The ability to generate precursor-product ion tables from this information and subsequently identify the correct parent precursor peptide will be illustrated in a companion manuscript.
We carried out a test sample study to try to identify errors leading to irreproducibility, including incompleteness of peptide sampling, in LC-MS-based proteomics. We distributed a test sample consisting of an equimolar mix of 20 highly purified recombinant human proteins, to 27 laboratories for identification. Each protein contained one or more unique tryptic peptides of 1250 Da to also test for ion selection and sampling in the mass spectrometer. Of the 27 labs, initially only 7 labs reported all 20 proteins correctly, and only 1 lab reported all the tryptic peptides of 1250 Da. Nevertheless, a subsequent centralized analysis of the raw data revealed that all 20 proteins and most of the 1250 Da peptides had in fact been detected by all 27 labs. The centralized analysis allowed us to determine sources of problems encountered in the study, which include missed identifications (false negatives), environmental contamination, database matching, and curation of protein identifications. Improved search engines and databases are likely to increase the fidelity of mass spectrometry-based proteomics.
We describe a novel LCMS approach to the relative quantitation and simultaneous identification of proteins within the complex milieu of unfractionated Escherichia coli. This label-free, LCMS acquisition method observes all detectable, eluting peptides and their corresponding fragment ions. Postacquisition data analysis methods extract both the chromatographic and the mass spectrometric information on the tryptic peptides to provide time-resolved, accurate mass measurements, which are subsequently used for quantitation and identification of constituent proteins. The response of E. coli to carbon source variation is well understood, and it is thus commonly used as a model biological system when validating an analytical method. Using this LCMS approach, we characterized proteins isolated from E. coli grown in glucose, lactose, and acetate. The change in relative abundance of the corresponding proteins was measured from peptides common to both conditions. Protein identities were also determined for those peptides that were unique to each condition, and these identities were found to be consistent with the underlying biochemical restrictions imposed by the growth conditions. The relative change in abundance of the characterized proteins ranged from 0.1-to 90-fold among the three binary comparisons. The overall coverage of the characterized proteins ranged from 10 to Escherichia coli is a microbial symbiote found in the colon and large intestine of most warm blooded animals that plays a critical role in vertebrate anabolism and catabolism. The environment in which E. coli lives is subject to rapid changes in the availability of the carbon and nitrogen compounds necessary to provide its energy and primary building blocks. E. coli survival hinges on the ability to successfully control the expression of genes coding for enzymes and proteins required for growth in response to environmental changes. Because of its simple cellular structure and its relative ease of maintenance and manipulation in the laboratory, E. coli has become the "workhorse host" for most research in molecular biology and microbiology. As a result, it is regarded as one of the most completely characterized organisms in all biology. The ease with which recombinant proteins can be expressed in E. coli has made this bacterium useful in the study of many basic biological processes as well as in the production of heterologous proteins for research and therapeutic purposes. For these reasons, E. coli has become a model system for testing new analytical technologies. For example, the relatively small genome size and prevalent laboratory use made E. coli genome one of the first to be completely sequenced (1). Likewise E. coli genome microarrays were among the first to be commercially available with sequences for the complete set of open reading frames as well as intergenic regions (2). The origins of proteomics can also be traced back to E. coli when pioneering two-dimensional gel electrophoresis experiments enabled the investigation of proteins on an organism...
A study of processed peanut oil was undertaken to assess the utility of HPLC combined with tandem MS to obtain data easily regarding the number of TAG of fats and oils and their FA composition. Mass chromatograms and spectra corresponding to only TAG of a single M.W. were obtained for the full range of TAG in the sample. Analysis of the mass spectra allowed the identification of more than 160 TAG in the sample by their FA composition. In addition, it was possible to estimate relative abundances of the TAG and suggest the position of the FA on glycerol for a limited number of cases. This technique greatly simplifies the task of assigning FA to coeluting TAG and facilitates identification of TAG present in trace quantities in mixtures, with possible application in circumstances where such trace TAG could be significant markers. Results are quickly obtained without extensive sample preparation or prefractionation of the sample.Paper no. J10065 in JAOCS 79, 749-753 (August 2002).Separation and identification of components of the complex mixtures of TAG that constitute fats and oils is a challenging analytical chemistry problem. Various approaches to separation by HPLC have been studied over the years (1). MS, and in particular combined HPLC and MS, has substantially improved the analysis of these mixtures (2). Relatively little has been reported concerning the use of electrospray ionization (ESI) MS, ESI-LC-MS, and tandem MS (MS-MS) in the analysis of TAG mixtures. Duffin et al. (3) first reported ESI-MS-MS analysis of TAG (as ammonium or sodium ion adducts) by infusing a solution of the analytes in nonpolar solvents. Studies leading to complete structures of individual TAG by ESI or fast atom bombardment MS-MS on tandem magnetic sector instruments were undertaken by Cheng et al. (4). Hsu and Turk (5) described the use of ESI-MS-MS in a triple quadrupole instrument for the structural elucidation of lithium ion adducts of individual TAG. Marzilli and coworkers (6) studied the selectivity of FA loss from sn-1,3 vs. sn-2 positions in a small set of TAG under MS-MS and MS 3 conditions in an ion trap mass spectrometer.In a preliminary study for this paper, the FA composition of TAG components of cocoa butter was studied by ESI-MS-MS (7).Peanut oil was chosen as a test substance for the ability of LC-MS-MS to identify TAG components because it contains a wider range of FA (from at least C 16 to C 26 ) than is typical for vegetable oils. Previous reports on the characterization of peanut oil have involved combinations of analytical methods. Myher et al. (8) analyzed natural and randomized peanut oils by HPLC coupled to direct inlet chemical ionization MS, identifying 35 TAG molecular species in a randomized sample and 16 TAG molecular species in natural samples. Sempore and Bezard (9) collected fractions from isocratic HPLC with refractive index detection, analyzed the FA of the fractions as methyl esters by GLC, and deduced the composition of the peanut oil TAG based on parameters derived from retention time data and mathe...
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