Dataset Information

Online alkaline-pH reverse phase nanoelectrospray-tandem mass spectrometry complements conventional low-pH method for global proteomic analysis

ABSTRACT: The global proteome analysis was limited by the identification of peptides with low abundance or specific physiochemical properties. Here, a one-dimensional online alkaline-pH reverse phase nanoelectrospray-tandem mass spectrometry (AlkalinepH-MS/MS) method was developed and optimized for global proteomic analysis. In this method, peptides were separated on a nanoflow C18 column with an alkaline-pH mobile phase (pH=8) and directly introduced to the mass spectrometer through nanoelectrospray ionization. The identified peptides overlapped between AlkalinepH-MS/MS and conventional online low-pH reverse phase nanoelectrospray-tandem mass spectrometry (LowpH-MS/MS) were as low as 45%, indicating that these two methods were complementary to each other. In addition, the AlkalinepH-MS/MS method showed identification capacity for a higher proportion of peptides with low molecular weight (MW), positive grand average of hydropathy (GRAVY), or high isoelectric point (pI). Moreover, the AlkalinepH-MS/MS method had a bias toward histidine, lysine, methionine, and proline- containing peptides. The complementarity of AlkalinepH-MS/MS and LowpH-MS/MS was further demonstrated for the analysis of tryptic digests from 15 intrahepatic cholangiocarcinoma (iCCA) cell lines. The alternating of 60-min AlkalinepH-MS/MS and 60-min LowpH-MS/MS method outperformed the conventional 120-min LowpH-MS/MS method in the identification of amino acid variants and protein groups. Our AlkalinepH-MS/MS method was an excellent complementary and alternative method to the LowpH-MS/MS method for global proteomic analysis.

ORGANISM(S): Homo Sapiens

SUBMITTER: Hu Zhou

PROVIDER: PXD039453 | iProX | Sun Jan 15 00:00:00 GMT 2023

REPOSITORIES: iProX

ACCESS DATA

Publications

Online Alkaline-pH Reverse Phase Nanoelectrospray-Tandem Mass Spectrometry Complements Conventional Low-pH Method for Global Proteomic Analysis.

Gao Jing J Wang Yuqiu Y Zhang Shu S Yang Zhicheng Z Zhu Hongwen H Luo Xuanyang X Lu Dayun D Gao Qiang Q Zhou Hu H

Journal of proteome research 20230207 5

The global proteome analysis was limited by the identification of peptides with low abundance or specific physiochemical properties. Here, a one-dimensional online alkaline-pH reverse phase nanoelectrospray-tandem mass spectrometry (alkaline-pH-MS/MS) method was developed and optimized for global proteomic analysis. In this method, peptides were separated on a nanoflow C18 column with an alkaline-pH mobile phase (pH = 8.0) and directly injected into the mass spectrometer. The unique peptides ove ...[more]

PMID: 36751022

Similar Datasets

Project description:Exposure of Saccharomyces cerevisiae to alkaline pH represents a stress condition that generates a compensatory reaction. Here we examine a possible role of the protein kinase-A (PKA) pathway in this response. The phenotypic analysis reveals that mutations that activate the PKA pathway (ira1 ira2, bcy1) tend to cause sensitivity to alkaline pH, whereas its deactivation develops tolerance to this stress. We observe that alkalinization causes a transient decrease in cAMP, the main regulator of the pathway. Alkaline pH causes rapid nuclear localization of the PKA-regulated Msn2 transcription factor which, together with Msn4, mediates a general stress response by binding to STRE sequences in many promoters. Consequently, a synthetic STRE-LacZ reporter shows a rapid induction in response to alkaline stress. An msn2 msn4 mutant is sensitive to alkaline pH, and transcriptomic analysis reveals that after 10 minutes of alkaline stress, the expression of many induced genes (47%) depends, at least in part, on the presence of Msn2 and Msn4. Taken together, these results demonstrate that inhibition of the PKA pathway by alkaline pH represents a substantial part of the adaptive response to this kind of stress and that this response involves Msn2/Msn4-mediated gene remodeling. However, the relevance of attenuation of PKA in high pH tolerance is not restricted to regulation of Msn2 function. Eight samples were analyzed: WT and the MCY5278 mutant strain, lacking both Msn2 and Msn4, in the presence of 20 mM KOH (pH 8) and in the presence of 20 mM KCl (non-induced conditions) for 10 and 30 min of stress. 2 biological replicates were analyzed for each condition, and dye-swapping was carried out for each comparison of samples. We compared the expression profiles of: 1) WT +KOH vs. WT +KCl after 10 min 2) msn2 msn4 mutant +KOH vs. msn2 msn4 +KCl after 10 min 3)WT +KOH vs. WT +KCl after 30 min 4) msn2 msn4 mutant +KOH vs. msn2 msn4 +KCl after 30 min Total number of chips analyzed: 16.

Project description:【Objective】 The objective of this study is to study the transcriptome regulation mechanism of F. graminearum under different pH stress conditions, analyze the gene expression level and its differences, and explore the metabolic pathways related to the anti-stress response of F. graminearum cells under acidic or alkaline conditions, and reveal how F. graminearum actively regulates intracellular metabolism and synthesis processes to adapt to the changes of extracellular pH environment. 【Method】 F. graminearum was cultured in PDB (potato dextrose broth) medium with initial pH of 4.5, 6.5 and 8.0 for 48 h, and the total RNA of the strain was extracted to construct a cDNA library. Transcriptome sequencing and bioinformatics techniques were used to identify the related differentially expressed genes (DEGs), and the metabolic pathways involved were further analyzed. 【Result】 A total of 4283 DEGs were detected under acidic conditions, of which 2232 were up-regulated and 2252 were down-regulated. Under alkaline conditions, there were a total of 498 DEGs, of which 269 were up-regulated and 229 were down-regulated. The results of Gene Ontology (GO) functional enrichment analysis showed that 211 GO terms were significantly enriched and 72 were down-regulated under acidic conditions. There were 33 GO terms that were revised upwards and 40 downwards under alkaline conditions. The results of KEGG encyclopedia of genes and Genomes (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis showed that 22 pathways were significantly enriched and 32 pathways were down-regulated under acidic conditions. There were 8 up-regulated pathways and 13 down-regulated pathways under alkaline conditions. The expression of membrane transporters and hydrolysis of carbohydrate compounds and other related genes were up-regulated, and the expression of genes related to protein metabolism was down-regulated, which assisted F. graminearum cells to adapt to changes in the external environment. At the same time, F. graminearum maintained the internal environment balance of its own cells by reducing secondary metabolism and amino acid metabolism under acidic and alkaline conditions, respectively, so as to resist extracellular pH stress. 【Conclusion】 In the acidic environment, Fusarium graminearum adapts to the changes in the extracellular environment by promoting the production of riboprotein complexes and secondary metabolism. In an alkaline environment, Fusarium graminearum cells respond to and sense external stresses through amino acid metabolism. The analysis of the metabolic pathways of F. graminearum cells provides important gene expression data for the response of F. graminearum to different pH environments, and the results of this study are helpful to understand the pathogenesis of F. graminearum.

Project description:Staphylococcus aureus is a leading cause of hospital- and community-associated infections. The organism’s ability to cause disease can, in part, be attributable to its ability to adapt to otherwise deleterious host-associated stresses. Like other bacterial species, the modulation of mRNA turnover appears to play an important role in S. aureus adaptation to certain environmental stresses. In the current study Affymetrix GeneChips® were used to examine the S. aureus responses to acid and alkaline shock-inducing conditions and to assess whether stress dependent changes in mRNA turnover are likely to facilitate the organism’s ability to tolerate extreme pH challenge. Results indicate that S. aureus adapts to pH shock by eliciting responses expected of organisms coping with pH alteration, including neutralizing cellular internal pH, DNA repair, amino acid biosynthesis and virulence factor expression. Further, it was found that the cellular response to alkaline conditions elicits a transcriptional profile that is similar to that of stringent response induced cells. Consistent with that observation, we show that the activator of the stringent response, (p)ppGpp, levels are profoundly elevated during alkaline shock conditions. We also show that the mRNA turnover properties of acid or alkaline shocked cells significantly differ from that of cells grown at neutral pH. A comparison of the mRNA degradation properties of transcripts whose titers either increased or decreased in response to sudden pH change revealed that alterations in mRNA degradation may, in part, account for the changes in the mRNA levels of factors predicted to mediate pH tolerance. Finally, a set of small stable RNA molecules were induced in response to acid or alkaline shock conditions. As in other organisms, these molecules may mediate mRNA stability and adaptation to otherwise deleterious growth conditions. Staphylococcus aureus strain UAMS-1 was grown to exponential phase and either mock treated (pH maintained at 7.4) or subjected to acid (pH 4)- or alkaline (pH 10) conditions for 30 min. Next, 200 micrograms per ml of rifampicin were added to arrest transcript synthesis. RNA was extracted from cell suspensions at 0, 2.5, 5, 15, and 30 min post-transcriptional arrest, labeled and hybridized to S. aureus GeneChips. A comparison of 0 min samples allowed assessment of acid and alkaline shock responses. A comparison of 0 min to that of various post-transcriptional arrest RNA samples allowed assessment of the mRNA turnover properties of mock vs acid or alkaline shocked cells. Duplicates of each experimental condition and corresponding post-transcriptional arrest time point were used (biological replicates).

Dataset Information

Online alkaline-pH reverse phase nanoelectrospray-tandem mass spectrometry complements conventional low-pH method for global proteomic analysis

Publications

Online Alkaline-pH Reverse Phase Nanoelectrospray-Tandem Mass Spectrometry Complements Conventional Low-pH Method for Global Proteomic Analysis.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets