Protein and peptide fractionation, enrichment and depletion: Tools for the complex proteome

Ly, Linda; Wasinger, Valerie C.

doi:10.1002/pmic.201000394

Cited by 91 publications

(69 citation statements)

References 220 publications

(233 reference statements)

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“…Minor constituents of highly complex mixtures of tryptic peptides (such as the trypsinized "shotgun" proteome of Hfx. volcanii) are often not readily identified by MS-based technologies (57). Thus, we suggest that at least a portion of the proteome associated with SAMP3 in a UbaA-dependent manner is of low abundance in the cell.…”

Section: Samp3ylation Sites Identified Via Lc-ms/ms Proteomic Analysismentioning

confidence: 87%

Archaeal Ubiquitin-like SAMP3 is Isopeptide-linked to Proteins via a UbaA-dependent Mechanism

Miranda

Antelmann

Hepowit

et al. 2014

Molecular & Cellular Proteomics

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SAMP1 and SAMP2 are ubiquitin-like proteins that function as protein modifiers and are required for the production of sulfur-containing biomolecules in the archaeon Haloferax volcanii. Here we report a novel small archaeal modifier protein (named SAMP3) with a ␤-grasp fold and C-terminal diglycine motif characteristic of ubiquitin that is functional in protein conjugation in Hfx. volcanii. SAMP3 conjugates were dependent on the ubiquitin-activating E1 enzyme homolog of archaea (UbaA) for synthesis and were cleaved by the JAMM/MPN؉ domain metalloprotease HvJAMM1. Twenty-three proteins (28 lysine residues) were found to be isopeptide-linked to the C-terminal carboxylate of SAMP3, and 331 proteins were reproducibly found associated with SAMP3 in a UbaA-dependent manner based on tandem mass spectrometry (MS/MS) analysis. The molybdopterin (MPT) synthase large subunit homolog MoaE, found samp3ylated at conserved active site lysine residues in MS/MS analysis, was also shown to be covalently bound to SAMP3 by immunoprecipitation and tandem affinity purifications. HvJAMM1 was demonstrated to catalyze the cleavage of SAMP3 from MoaE, suggesting a mechanism of controlling MPT synthase activity. The levels of samp3ylated proteins and samp3 transcripts were found to be increased by the addition of dimethyl sulfoxide to aerobically growing cells. Thus, we propose a model in which samp3ylation is covalent and reversible and controls the activity of enzymes such as MPT synthase. Sampylation of MPT synthase may govern the levels of molybdenum cofactor available and thus facilitate the scavenging of oxygen prior to the transition to respiration with molybdenum-cofactor-containing terminal reductases that use alternative electron acceptors such as dimethyl sulfoxide. Overall, our study of SAMP3 provides new insight into the diversity of functional ubiquitin-like protein modifiers and the network of ubiquitin-like protein targets in Archaea. Molecular & Cellular

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Section: Samp3ylation Sites Identified Via Lc-ms/ms Proteomic Analysismentioning

confidence: 87%

Archaeal Ubiquitin-like SAMP3 is Isopeptide-linked to Proteins via a UbaA-dependent Mechanism

Miranda

Antelmann

Hepowit

et al. 2014

Molecular & Cellular Proteomics

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“…They improve the efficiency of MS-based protein identification and allow the association of different sets of proteins to specific cell compartments [17]. The carried out fractioning approach allowed to generate soluble fractions, enriched with cytoplasmic proteins (CyPE), and insoluble fractions, enriched with surface proteins (SuPE).…”

Section: Discussionmentioning

confidence: 99%

Comparative proteomics of two Mycoplasma hyopneumoniae strains and Mycoplasma flocculare identified potential porcine enzootic pneumonia determinants

et al. 2018

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Mycoplasma hyopneumoniae and Mycoplasma flocculare are genetically similar bacteria, which coinhabit the porcine respiratory tract. These mycoplasmas share most of the known virulence factors, but, while M. hyopneumoniae causes porcine enzootic pneumonia (PEP), M. flocculare is a commensal species. To identify potential PEP determinants and provide novel insights on mycoplasma-host interactions, the whole cell proteomes of two M. hyopneumoniae strains, one pathogenic (7448) and other non-pathogenic (J), and M. flocculare were compared. A cell fractioning approach combined with mass spectrometry (LC-MS/MS) proteomics was used to analyze cytoplasmic and surface-enriched protein fractions. Average detection of ~ 50% of the predicted proteomes of M. hyopneumoniae 7448 and J, and M. flocculare was achieved. Many of the identified proteins were differentially represented in M. hyopneumoniae 7448 in comparison to M. hyopneumoniae J and M. flocculare, including potential PEP determinants, such as adhesins, proteases, and redox-balancing proteins, among others. The LC-MS/MS data also provided experimental validation for several genes previously regarded as hypothetical for all analyzed mycoplasmas, including some coding for proteins bearing virulence-related functional domains. The comprehensive proteome profiling of two M. hyopneumoniae strains and M. flocculare provided tens of novel candidates to PEP determinants or virulence factors, beyond those classically described.

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“…Larger particle size phases form the basis of low-pressure liquid chromatography in which the flow of eluent through the column is either gravity-fed or pumped by a low pressure pump, often a peristaltic pump. This technique separates proteins through a bed of porous beads where the molecules are eluted off the column in order of decreasing size [49].…”

Section: High-performance Liquid Chromatography (Hpcl)mentioning

confidence: 99%

Musculoskeletal ProteinAnalysis Techniques - A Review

Kamineni¹,

Manepally²,

Kamineni³

2016

JRAD

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There have been a large number of experimental methods for purifying and analyzing proteins from the sample of interest. Determination of protein concentration is often the key step and is common to many applications in protein research and life sciences. The most commonly used quantitative techniques to measure the protein concentration are Amino acid analysis, Biuret assay, Bradford assays, Folin-Lowry assay, Bicinchoninic (BCA) assays, UV absorbance assays and Antibody-based assays such as ELISA and Western Blotting. Some of the qualitative methods that are used to detect different types of proteins and amino acids are Ninhydrin test, Xanthoproteic test, Millon's test, Sulfur test, Hopkins-Cole test, and Nitroprusside test. Chromatographic analysis can also be carried out on an either qualitative or quantitative basis to purify complex protein mixtures based on their properties such as size, solubility, charge, hydrophobicity and bio-specific interaction. Selection of these assays is based upon the ease of performance, range of concentrations, sensitivity, and interfering substances contained in the complex mixture. The purpose of this review is to provide an assessment of commonly used chromatographic and colorimetric methods for the determination of protein concentration.

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Protein and peptide fractionation, enrichment and depletion: Tools for the complex proteome

Cited by 91 publications

References 220 publications

Archaeal Ubiquitin-like SAMP3 is Isopeptide-linked to Proteins via a UbaA-dependent Mechanism

Archaeal Ubiquitin-like SAMP3 is Isopeptide-linked to Proteins via a UbaA-dependent Mechanism

Comparative proteomics of two Mycoplasma hyopneumoniae strains and Mycoplasma flocculare identified potential porcine enzootic pneumonia determinants

Musculoskeletal ProteinAnalysis Techniques - A Review

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