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      Quantitative proteomic analysis of single pancreatic islets

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          Abstract

          Technological developments make mass spectrometry (MS)-based proteomics a central pillar of biochemical research. MS has been very successful in cell culture systems, where sample amounts are not limiting. To extend its capabilities to extremely small, physiologically distinct cell types isolated from tissue, we developed a high sensitivity chromatographic system that measures nanogram protein mixtures for 8 h with very high resolution. This technology is based on splitting gradient effluents into a capture capillary and provides an inherent technical replicate. In a single analysis, this allowed us to characterize kidney glomeruli isolated by laser capture microdissection to a depth of more than 2,400 proteins. From pooled pancreatic islets of Langerhans, another type of "miniorgan," we obtained an in-depth proteome of 6,873 proteins, many of them involved in diabetes. We quantitatively compared the proteome of single islets, containing 2,000-4,000 cells, treated with high or low glucose levels, and covered most of the characteristic functions of beta cells. Our ultrasensitive analysis recapitulated known hyperglycemic changes but we also find components up-regulated such as the mitochondrial stress regulator Park7. Direct proteomic analysis of functionally distinct cellular structures opens up perspectives in physiology and pathology.

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          Most cited references 40

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          MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.

          Efficient analysis of very large amounts of raw data for peptide identification and protein quantification is a principal challenge in mass spectrometry (MS)-based proteomics. Here we describe MaxQuant, an integrated suite of algorithms specifically developed for high-resolution, quantitative MS data. Using correlation analysis and graph theory, MaxQuant detects peaks, isotope clusters and stable amino acid isotope-labeled (SILAC) peptide pairs as three-dimensional objects in m/z, elution time and signal intensity space. By integrating multiple mass measurements and correcting for linear and nonlinear mass offsets, we achieve mass accuracy in the p.p.b. range, a sixfold increase over standard techniques. We increase the proportion of identified fragmentation spectra to 73% for SILAC peptide pairs via unambiguous assignment of isotope and missed-cleavage state and individual mass precision. MaxQuant automatically quantifies several hundred thousand peptides per SILAC-proteome experiment and allows statistically robust identification and quantification of >4,000 proteins in mammalian cell lysates.
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            Mass spectrometry-based proteomics.

            Recent successes illustrate the role of mass spectrometry-based proteomics as an indispensable tool for molecular and cellular biology and for the emerging field of systems biology. These include the study of protein-protein interactions via affinity-based isolations on a small and proteome-wide scale, the mapping of numerous organelles, the concurrent description of the malaria parasite genome and proteome, and the generation of quantitative protein profiles from diverse species. The ability of mass spectrometry to identify and, increasingly, to precisely quantify thousands of proteins from complex samples can be expected to impact broadly on biology and medicine.
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              Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics.

              Proteomics is critically dependent on optimal sample preparation. Particularly, the interface between protein digestion and mass spectrometric analysis has a large influence on the overall quality and sensitivity of the analysis. We here describe a novel procedure in which a very small disk of beads embedded in a Teflon meshwork is placed as a microcolumn into pipet tips. Termed Stage, for STop And Go Extraction, the procedure has been implemented with commercially available material (C18 Empore Disks (3M, Minneapolis, MN)) as frit and separation material. The disk is introduced in a simple and fast process yielding a convenient and completely reliable procedure for the production of self-packed microcolumns in pipet tips. It is held in place free of obstacles solely by the narrowing tip, ensuring optimized loading and elution of analytes. Five disks are conveniently placed in 1 min, adding 300 micro/min for the packed column using manual force) while eliminating the possibility of blocking. The loading capacity of C18-StageTips (column bed: 0.4 mm diameter, 0.5 mm length) is 2-4 microg of protein digest, which can be increased by using larger diameter or stacked disks. Five femtomole of tryptic BSA digest could be recovered quantitatively. We have found that the Stage system is well-suited as a universal sample preparation system for proteomics.
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                Author and article information

                Journal
                Proceedings of the National Academy of Sciences
                PNAS
                Proceedings of the National Academy of Sciences
                0027-8424
                1091-6490
                November 10 2009
                November 10 2009
                November 10 2009
                October 21 2009
                : 106
                : 45
                : 18902-18907
                10.1073/pnas.0908351106
                2765458
                19846766
                © 2009

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