Comparison of ultrafiltration units for proteomic and N-glycoproteomic analysis by the filter-aided sample preparation method

Wiśniewski, Jacek R.; Zielińska, Dorota; Mann, Matthias

doi:10.1016/j.ab.2010.12.004

Cited by 166 publications

(163 citation statements)

References 20 publications

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“…In this method, molecular weight cutoff (MWCO) filters were used to remove any detergents and salts used for the affinity purification, which allows the flexible use of detergents and buffers without interfering with subsequent MS analyses. In the current protocol, the key features modified with respect to the previously published methods (26,27) include the use of trifluoroethanol (TFE) in addition to urea for solublization of the protein samples and the substitution of Tris(2-carboxyethyl)phosphine (TCEP) for DTT in protein reduction. The use of TFE in place of or in addition to urea alone results in improved protein coverage by LC-MS in this experiment.…”

Section: Resultsmentioning

confidence: 99%

“…To prepare samples for MS, the eluent samples from Csy4* affinity purification were rinsed twice in 8 M urea using MWCO filters, followed by the addition of 40% TFE for efficient denaturation of the target proteins, allowing the effective removal even of detergents with low critical micelle concentrations, such as Triton X-100 (27). After the reduction of disulfide bonds between cysteine residues with TCEP and alkylation of free thiol groups with iodoacetamide to prevent disulfide bond formation during downstream peptide mapping by MS, the proteins were digested in two steps with endoproteinase LysC and then with trypsin.…”

Section: Resultsmentioning

confidence: 99%

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RNA–protein analysis using a conditional CRISPR nuclease

Lee

Haurwitz

Apffel

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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RNA-binding proteins control the fate and function of the transcriptome in all cells. Here we present technology for isolating RNA-protein partners efficiently and accurately using an engineered clustered regularly interspaced short palindromic repeats (CRISPR) endoribonuclease. An inactive version of the Csy4 nuclease binds irreversibly to transcripts engineered with a 16-nt hairpin sequence at their 5′ ends. Once immobilized by Csy4 on a solid support, contaminating proteins and other molecules can be removed by extensive washing. Upon addition of imidazole, Csy4 is activated to cleave the RNA, removing the hairpin tag and releasing the native transcript along with its specifically bound protein partners. This conditional Csy4 enzyme enables recovery of specific RNA-binding partners with minimal false-positive contamination. We use this method, coupled with quantitative MS, to identify cell type-specific human pre-microRNA-binding proteins. We also show that this technology is suitable for analyzing diverse size transcripts, and that it is suitable for adaptation to a high-throughput discovery format.non-coding RNA | RNA processing | miRNA | mass spectrometry R NA molecules function together with specific binding proteins to regulate cellular pathways at the levels of transcription, posttranscriptional modification, and translation (1-4). For example, microRNAs (miRNA), siRNAs, and piwi-interacting RNAs (piRNAs) regulate more than 30% of mammalian gene expression (5). Small nucleolar RNAs (snoRNAs) govern the sites and efficiencies of RNA chemical modifications in cells (6), whereas long noncoding RNAs (lncRNAs), such as HOTAIR and MALAT1, have been implicated in chromatin remodeling, transcriptional activation, and tumorigenesis (7,8). UTRs of mRNA transcripts are also known to interact with regulatory proteins to control their expression level as well as their stability. Understanding how these RNAs function in cells and how they may be manipulated for therapeutic purposes is an important goal that spans many areas of biology.Although new ncRNAs are being discovered at a rapid pace, determination of their biochemical activities is often slow. A major barrier to such functional analysis is the current difficulty of identifying RNA-binding partners that associate with specific transcripts and participate in their biological behavior. Despite the development of various RNA affinity purification methods, the expense and/or technical challenges associated with each approach has precluded its use by nonspecialists or in high-throughput discovery experiments. Current strategies for identifying RNA-binding proteins that associate with specific transcripts involve the use of affinity tags, including biotin, aptamers, and particular proteinbinding sequences (9-13). In each case, however, the modest affinity or specificity of tag recognition and the difficulty of selective elution complicate sample analysis. A strategy that will allow simple and rapid identification of proteins that associate selectively with particu...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

RNA–protein analysis using a conditional CRISPR nuclease

Lee

Haurwitz

Apffel

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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“…Supernatants were diluted with buffer UA (8 M urea, 0.1 M Tris/HCl pH ϭ 8.5) to a final concentration of 0.5% SDS. Proteins were digested with the endoproteinase LysC following the protocol for filter aided sample preparation (18). Briefly, protein extracts were loaded on a 30k Amicon Ultra filter unit (Millipore, Billerica, MA) by means of centrifugation at 14,000g.…”

Section: Methodsmentioning

confidence: 99%

Native SILAC: Metabolic Labeling of Proteins in Prototroph Microorganisms Based on Lysine Synthesis Regulation

Fröhlich

Christiano

Walther

2013

Molecular & Cellular Proteomics

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Mass spectrometry (MS)-based quantitative proteomics has matured into a methodology able to detect and quantitate essentially all proteins of model microorganisms, allowing for unprecedented depth in systematic protein analyses. The most accurate quantitation approaches currently require lysine auxotrophic strains, which precludes analysis of most existing mutants, strain collections, or commercially important strains (e.g. those used for brewing or for the biotechnological production of metabolites). Here, we used MS-based proteomics to determine the global response of prototrophic yeast and bacteria to exogenous lysine. Unexpectedly, down-regulation of lysine synthesis in the presence of exogenous lysine is achieved via different mechanisms in different yeast strains. In each case, however, lysine in the medium down-regulates its biosynthesis, allowing for metabolic proteome labeling with heavy-isotope-containing lysine. This strategy of native stable isotope labeling by amino acids in cell culture (nSILAC) overcomes the limitations of previous approaches and can be used for the efficient production of protein standards for absolute SILAC quantitation in model microorganisms. As proof of principle, we have used nSILAC to globally analyze yeast proteome changes during salt stress. Molecular & Cellular Proteomics 12:

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“…The Suc gradient was fractionated, and protein concentrations of peak fractions of LHCII monomers, LHCII trimers, PSII core complexes, PSI-LHCI, PSII-LHCII, and supercomplexes were determined using the Pierce BCA Protein Assay Kit (Thermo Scientific) according to the manufacturer's instructions. Samples were tryptically digested in 0.5-mL Amicon Ultra ultrafiltration devices (30-kD cutoff; Millipore) according to the FASP method (Wi sniewski et al, 2009(Wi sniewski et al, , 2011 with minor modifications. Proteins were reduced with 100 mM dithiothreitol in 100 mM Tris-HCl (pH 8.5) and 8 M urea (UA) at room temperature for 30 min.…”

Section: Tryptic Digestion Of Thylakoid Membrane Proteinsmentioning

confidence: 99%

STATE TRANSITION7-Dependent Phosphorylation Is Modulated by Changing Environmental Conditions, and Its Absence Triggers Remodeling of Photosynthetic Protein Complexes

et al. 2015

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In plants and algae, the serine/threonine kinase STN7/STT7, orthologous protein kinases in Chlamydomonas reinhardtii and Arabidopsis (Arabidopsis thaliana), respectively, is an important regulator in acclimation to changing light environments. In this work, we assessed STT7-dependent protein phosphorylation under high light in C. reinhardtii, known to fully induce the expression of LIGHT-HARVESTING COMPLEX STRESS-RELATED PROTEIN3 (LHCSR3) and a nonphotochemical quenching mechanism, in relationship to anoxia where the activity of cyclic electron flow is stimulated. Our quantitative proteomics data revealed numerous unique STT7 protein substrates and STT7-dependent protein phosphorylation variations that were reliant on the environmental condition. These results indicate that STT7-dependent phosphorylation is modulated by the environment and point to an intricate chloroplast phosphorylation network responding in a highly sensitive and dynamic manner to environmental cues and alterations in kinase function. Functionally, the absence of the STT7 kinase triggered changes in protein expression and photoinhibition of photosystem I (PSI) and resulted in the remodeling of photosynthetic complexes. This remodeling initiated a pronounced association of LHCSR3 with PSI-LIGHT HARVESTING COMPLEX I (LHCI)-ferredoxin-NADPH oxidoreductase supercomplexes. Lack of STT7 kinase strongly diminished PSII-LHCII supercomplexes, while PSII core complex phosphorylation and accumulation were significantly enhanced. In conclusion, our study provides strong evidence that the regulation of protein phosphorylation is critical for driving successful acclimation to high light and anoxic growth environments and gives new insights into acclimation strategies to these environmental conditions. Oxygenic photosynthesis converts solar energy into chemical energy. This energy is utilized for carbon dioxide assimilation, allowing the formation of complex organic material. Plant photosynthesis is performed by a series of reactions in and at the thylakoid membrane, resulting in light-dependent water oxidation, NADP reduction, and ATP formation (Whatley et al., 1963). These light reactions are catalyzed by two photosystems (PSI and PSII). A third multiprotein complex, also embedded in the thylakoid membrane, is the cytochrome b 6 f (cyt b 6 f) complex that links photosynthetic electron transfer processes between the two photosystems and functions in proton translocation. The ATP synthase takes advantage of the proton-motive force that is generated by the light reactions (Mitchell, 1961) to produce ATP. ATP and NADPH, generated through linear electron flow from PSII to PSI, drive the CalvinBenson-Bassham cycle (Bassham et al., 1950) to fix CO 2 . Alternatively, cyclic electron flow (CEF) between PSI and the cyt b 6 f complex solely produces ATP (Arnon, 1959).Under normal growth conditions, CEF provides additionally required ATP for CO 2 fixation (Lucker and Kramer, 2013), counteracts overreduction of the PSI acceptor side under stressful environmental cues,...

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Comparison of ultrafiltration units for proteomic and N-glycoproteomic analysis by the filter-aided sample preparation method

Cited by 166 publications

References 20 publications

RNA–protein analysis using a conditional CRISPR nuclease

RNA–protein analysis using a conditional CRISPR nuclease

Native SILAC: Metabolic Labeling of Proteins in Prototroph Microorganisms Based on Lysine Synthesis Regulation

STATE TRANSITION7-Dependent Phosphorylation Is Modulated by Changing Environmental Conditions, and Its Absence Triggers Remodeling of Photosynthetic Protein Complexes

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