Peptide affinity analysis of proteins that bind to an unstructured region containing the transactivating domain of the osmoprotective transcription factor NFAT5.

NFAT5 is a transcription factor originally identified because it is activated by hypertonicity and that activation increases expression of genes that protect against the adverse effects of the hypertonicity. However, its targets also include genes not obviously related to tonicity. The transactivating domain of NFAT5 is contained in its COOH-terminal region, which is predicted to be unstructured. Unstructured regions are common in transcription factors particularly in transactivating domains where they can bind co-regulatory proteins essential to their function. To identify potential binding partners of NFAT5 from either cytoplasmic or nuclear HEK293 cell extracts, we used peptide affinity chromatography followed by mass spectrometry. Peptide aptamer-baits consisted of overlapping 20 amino acid peptides within the predicted COOH-terminal unstructured region of NFAT5. We identify a total of 351 unique protein preys that associate with at least one COOH-terminal peptide bait from NFAT5 in either cytoplasmic or nuclear extracts from cells incubated at various tonicities (NaCl varied). In addition to finding many proteins already known to associate with NFAT5, we found many new ones whose function suggest novel aspects of NFAT5 regulation, interaction, and function. Relatively few of the proteins pulled down by peptide baits from NFAT5 are generally involved in transcription, and most, therefore, are likely to be specifically related to the regulation of NFAT5 or its function. The novel associated proteins are involved with cancer, effects of hypertonicity on chromatin, development, splicing of mRNA, transcription, and vesicle trafficking.

Peptide affinity analysis of proteins that bind to an unstructured NH2-terminal region of the osmoprotective transcription factor NFAT5.

NFAT5 is an osmoregulated transcription factor that particularly increases expression of genes involved in protection against hypertonicity. Transcription factors often contain unstructured regions that bind co-regulatory proteins that are crucial for their function. The NH2-terminal region of NFAT5 contains regions predicted to be intrinsically disordered. We used peptide aptamer-based affinity chromatography coupled with mass spectrometry to identify protein preys pulled down by one or more overlapping 20 amino acid peptide baits within a predicted NH2-terminal unstructured region of NFAT5. We identify a total of 467 unique protein preys that associate with at least one NH2-terminal peptide bait from NFAT5 in either cytoplasmic or nuclear extracts from HEK293 cells treated with elevated, normal, or reduced NaCl concentrations. Different sets of proteins are pulled down from nuclear vs. cytoplasmic extracts. We used GeneCards to ascertain known functions of the protein preys. The protein preys include many that were previously known, but also many novel ones. Consideration of the novel ones suggests many aspects of NFAT5 regulation, interaction and function that were not previously appreciated, for example, hypertonicity inhibits NFAT5 by sumoylating it and the NFAT5 protein preys include components of the CHTOP complex that desumoylate proteins, an action that should contribute to activation of NFAT5.

Global discovery of high-NaCl-induced changes of protein phosphorylation.

High extracellular NaCl, such as in the renal medulla, can perturb and even kill cells, but cells mount protective responses that enable them to survive and function. Many high-NaCl-induced perturbations and protective responses are known, but the signaling pathways involved are less clear. Change in protein phosphorylation is a common mode of cell signaling, but there was no unbiased survey of protein phosphorylation in response to high NaCl. We used stable isotopic labeling of amino acids in cell culture coupled to mass spectrometry to identify changes in protein phosphorylation in human embryonic kidney (HEK 293) cells exposed to high NaCl. We reproducibly identify >8,000 unique phosphopeptides in 4 biological replicate samples with a 1% false discovery rate. High NaCl significantly changed phosphorylation of 253 proteins. Western analysis and targeted ion selection mass spectrometry confirm a representative sample of the phosphorylation events. We analyze the affected proteins by functional category to infer how altered protein phosphorylation might signal cellular responses to high NaCl, including alteration of cell cycle, cyto/nucleoskeletal organization, DNA double-strand breaks, transcription, proteostasis, metabolism of mRNA, and cell death.

A systematic quantitative proteomic examination of multidrug resistance in Acinetobacter baumannii.

Multidrug-resistant Acinetobacter baumannii strains have been examined at the DNA sequence level, but seldom using large-scale quantitative proteomics. We have compared the proteome of the multidrug resistant strain BAA-1605, with the proteome of the drug-sensitive strain ATCC 17978, using iTRAQ labeling and online 2D LC/MS/MS for peptide/protein identification. Of 1484 proteins present in at least 2 of 4 independent experiments, 114 are 2-fold to 66-fold more abundant in BAA-1605, and 99 are 2-fold to 50-fold less abundant. Proteins with 2-fold or greater abundance in the multidrug resistant strain include drug-, antibiotic-, and heavy metal-resistance proteins, stress-related proteins, porins, membrane transporters, proteins important for acquisition of foreign DNA, biofilm-related proteins, cell-wall and exopolysaccharide-related proteins, lipoproteins, metabolic proteins, and many with no annotated function. The porin CarO, inactivated in carbapenem-resistant strains, is 2.3-fold more abundant in BAA-1605. Likewise, the porin OmpW, less abundant in carbapenem- and colistin-resistant A. baumannii strains, is 3-fold more abundant in BAA-1605. Nine proteins, all present in the drug-sensitive strain but from 2.2-fold to 16-fold more abundant in the MDR strain, can potentially account for the observed resistance of BAA-1605 to 18 antibiotics. BIOLOGICAL SIGNIFICANCE: Multidrug resistant (MDR) strains of the pathogen Acinetobacter baumannii are a significant cause of hospital-acquired infections, are associated with increased mortality and length of stay, and may be a major factor underlying the spread of this pathogen, which is difficult to eradicate from clinical settings. To obtain a better understanding of antimicrobial resistance mechanisms in MDR A. baumannii, we report the first large scale 2D LC/MS/MS-based quantitative proteomics comparison of a drug-sensitive strain and an MDR strain of this pathogen. Ca. 20% of the expressed proteome changes 2-fold or more between the compared strains, including 42 proteins with literature or informatics annotations related to resistance mechanisms, modification of xenobiotics, or drug transport. Other categories of proteins differing 2-fold or more between strains include stress-response related proteins, porins, OMPs, transporters and secretion-related proteins, cell wall- and expolysaccharide-related proteins, lipoproteins, and DNA- and plasmid-related proteins. While the compared strains also differ in other aspects than multi-drug resistance, the observed differences, combined with protein functional annotation, suggest that complex protein expression changes may accompany the MDR phenotype. Expression changes of nine proteins in the MDR strain can potentially account for the observed resistance to 18 antibiotics.

Rapid identification of sequences for orphan enzymes to power accurate protein annotation.

The power of genome sequencing depends on the ability to understand what those genes and their proteins products actually do. The automated methods used to assign functions to putative proteins in newly sequenced organisms are limited by the size of our library of proteins with both known function and sequence. Unfortunately this library grows slowly, lagging well behind the rapid increase in novel protein sequences produced by modern genome sequencing methods. One potential source for rapidly expanding this functional library is the "back catalog" of enzymology--"orphan enzymes," those enzymes that have been characterized and yet lack any associated sequence. There are hundreds of orphan enzymes in the Enzyme Commission (EC) database alone. In this study, we demonstrate how this orphan enzyme "back catalog" is a fertile source for rapidly advancing the state of protein annotation. Starting from three orphan enzyme samples, we applied mass-spectrometry based analysis and computational methods (including sequence similarity networks, sequence and structural alignments, and operon context analysis) to rapidly identify the specific sequence for each orphan while avoiding the most time- and labor-intensive aspects of typical sequence identifications. We then used these three new sequences to more accurately predict the catalytic function of 385 previously uncharacterized or misannotated proteins. We expect that this kind of rapid sequence identification could be efficiently applied on a larger scale to make enzymology's "back catalog" another powerful tool to drive accurate genome annotation.

The development of ciprofloxacin resistance in Pseudomonas aeruginosa involves multiple response stages and multiple proteins.

Microbes have developed resistance to nearly every antibiotic, yet the steps leading to drug resistance remain unclear. Here we report a multistage process by which Pseudomonas aeruginosa acquires drug resistance following exposure to ciprofloxacin at levels ranging from 0.5× to 8× the initial MIC. In stage I, susceptible cells are killed en masse by the exposure. In stage II, a small, slow to nongrowing population survives antibiotic exposure that does not exhibit significantly increased resistance according to the MIC measure. In stage III, exhibited at 0.5× to 4× the MIC, a growing population emerges to reconstitute the population, and these cells display heritable increases in drug resistance of up to 50 times the original level. We studied the stage III cells by proteomic methods to uncover differences in the regulatory pathways that are involved in this phenotype, revealing upregulation of phosphorylation on two proteins, succinate-semialdehyde dehydrogenase (SSADH) and methylmalonate-semialdehyde dehydrogenase (MMSADH), and also revealing upregulation of a highly conserved protein of unknown function. Transposon disruption in the encoding genes for each of these targets substantially dampened the ability of cells to develop the stage III phenotype. Considering these results in combination with computational models of resistance and genomic sequencing results, we postulate that stage III heritable resistance develops from a combination of both genomic mutations and modulation of one or more preexisting cellular pathways.

High-accuracy peptide mass fingerprinting using peak intensity data with machine learning.

For MALDI-TOF mass spectrometry, we show that the intensity of a peptide-ion peak is directly correlated with its sequence, with the residues M, H, P, R, and L having the most substantial effect on ionization. We developed a machine learning approach that exploits this relationship to significantly improve peptide mass fingerprint (PMF) accuracy based on training data sets from both true-positive and false-positive PMF searches. The model's cross-validated accuracy in distinguishing real versus false-positive database search results is 91%, rivaling the accuracy of MS/MS-based protein identification.

Fragmentation characteristics of collision-induced dissociation in MALDI TOF/TOF mass spectrometry.

The identification of proteins by tandem mass spectrometry relies on knowledge of the products produced by collision-induced dissociation of peptide ions. Most previous work has focused on fragmentation statistics for ion trap systems. We analyzed fragmentation in MALDI TOF/TOF mass spectrometry, collecting statistics using a curated set of 2459 MS/MS spectra and applying bootstrap resampling to assess confidence intervals. We calculated the frequency of 18 product ion types, the correlation between both mass and intensity with ion type, the dependence of amide bond breakage on the residues surrounding the cleavage site, and the dependence of product ion detection on residues not adjacent to the cleavage site. The most frequently observed were internal ions, followed by y ions. A strong correlation between ion type and the mass and intensity of its peak was observed, with b and y ions producing the most intense and highest mass peaks. The amino acids P, W, D, and R had a strong effect on amide bond cleavage when situated next to the breakage site, whereas residues including I, K, and H had a strong effect on product ion observation when located in the peptide but not adjacent to the cleavage site, a novel observation.