How Much of the Proteome Do We See with Discovery-Based Proteomics Methods and How Much Do We Need to See?

Patterson, Scott D.

doi:10.2174/1570164043488306

Cited by 38 publications

(21 citation statements)

References 110 publications

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“…From our results, it is clear that even the 2-D ion trap mass spectrometer does not identify all or most of the analyzable peptides and also fails to identify all the peptides identified by the 3-D ion trap mass spectrometer. Estimating the complexity and then using suitable fractionation and enrichment techniques are more likely to provide biological leads and insights (19).…”

Section: Discussionmentioning

confidence: 99%

Systematic Comparison of a Two-dimensional Ion Trap and a Three-dimensional Ion Trap Mass Spectrometer in Proteomics

Mayya

Karim

Cong

et al. 2005

Molecular & Cellular Proteomics

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The utility and advantages of the recently introduced twodimensional 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 twodimensional ion trap over the conventional three-dimensional ion traps for protein identification in proteomic experiments. Molecular & Cellular Proteomics 4: 214 -223, 2005.Ion trap mass spectrometers are widely used in proteomic studies (1, 2). They are rugged, relatively less expensive, and provide good MS/MS 1 capabilities. A number of critical technological features also complement the utility of ion traps in proteomics. For example, software control of ion trap instrument operation has facilitated data-dependent acquisition features and, along with on-line reverse phase separation, has enabled analysis of highly complex peptide mixtures from cells and tissues (3-5). Furthermore search tools such as SEQUEST automate the process of assigning uninterpreted fragmentation data to peptides from sequence data bases, making proteomic scale projects feasible (6 -8).Despite a number of technological improvements, comprehensive identification of cellular proteomes is not fully realized. This limitation is due in part to the complexity of samples in proteomic experiments where thousands of peptides elute within a narrow window of organic gradient in reverse-phase separation methods (9). In addition, conventional three-dimensional (3-D) ion traps have a slower scan rate and a limited ion capacity and trapping efficiency (10). These may contribute toward a reduced number of peptides identified as well as limited quality of fragmentation spectra. Improving the above features of the ion trap mass spectrometers is likely to enhance the identification capability. To address these issues, a new design of the ion trap mass analyzer was introduced, and this is commercially available as LTQ (10). The LTQ has a quadrupole made of four hyperbolic cross-sectional rods giving it a two-dimensional (2-D) or linear feature. The ions are trapped in an axial fashion as opposed t...

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Section: Discussionmentioning

confidence: 99%

Systematic Comparison of a Two-dimensional Ion Trap and a Three-dimensional Ion Trap Mass Spectrometer in Proteomics

Mayya

Karim

Cong

et al. 2005

Molecular & Cellular Proteomics

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“…However, this information is already enough to perform valuable discovery-based science, and to increase our knowledge of these systems [89] . Starting from this position, two approaches may be envisaged: (1) a technological improvement, which would penetrate deeper into the system and increase the amount of data, and (2) the use of all the available data to discover emergent, or previously undiscovered, properties, apparently hidden by the complexity.…”

Section: Discussionmentioning

confidence: 99%

Challenges in the Overall Analysis of Microbial Proteomes

Scherl¹,

Sanchez²,

Hochstrasser³

2004

ComPlexUs

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Proteomics is widely used to analyze the protein content of bacterial species. Using various technologies, about 25–30% of the predicted proteins are typically identified. The relative abundance of these proteins at different stages, or under different conditions, can also be determined using proteomics approaches. In the first part of this review, a limited number of proteomics studies of bacterial species is summarized. This is not done in an exhaustive way, but to emphasize the complexity of the data which can be generated. Ways to manage and analyze this information are explored in the second part. The need to use standardized data formats, powerful bioinformatics tools and information management systems is discussed.

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“…The technique also needs to be suitable for downstream proteomics analysis procedures with minimal post-extraction artefacts and non-proteinaceous contaminants. The presence in plant cells of multiple interfering substances such as proteases, polyphenols, tannins, pigments, waxes, high carbohydrate/protein ratio further complicates the eventual extraction, solubilisation and separation procedures, that even under optimal conditions, results in the reduction of approximately 25% of the expected proteome (Patterson 2004). No single protein extraction protocol can capture an entire proteome, consequently a range of different extraction protocols, involving many permutations of physical and chemical treatments, solvent and buffers have been reported in literature (Rose et al 2004, Baginsky 2009.…”

Section: Protein Extractionmentioning

confidence: 99%

“…A challenge in comparative proteomics is the difficulty in delivering large-scale protein quantification to assay global protein changes elicited by biotic /abiotic events. A second problem is the inadequacy of current technologies for analysing a representative proportion of the expressed proteins present in a plant sample (Patterson 2004). This is mainly due to the dynamic range of protein concentrations within plant cells which is estimated to be as wide as 10 5 -10 6 (Pattersons & Aebersold 2003).…”

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confidence: 99%