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In-column thermostability of peptides

ABSTRACT: The primary objective of this study was to revise the advantages of temperature in peptide separations using state-of-the-art columns, but more importantly, to reveal possible mechanisms elevated temperature can impair proteomic bottom-up LC-MS analyses.

INSTRUMENT(S): Q Exactive HF-X

ORGANISM(S): Homo Sapiens (human) Francisella Tularensis Subsp. Holarctica (strain Lvs)

SUBMITTER: Juraj Lenco

LAB HEAD: Juraj Lenco, Ph.D.

PROVIDER: PXD019996 | Pride | 2020-11-17

REPOSITORIES: Pride

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			Action	DRS
	DataDescription_02.xlsx	Xlsx
	JL703_JL697_FtularensisLVS_Digest_2uGin1uL_0l5uL_000min_01.msf	Msf
	JL703_JL697_FtularensisLVS_Digest_2uGin1uL_0l5uL_000min_01.raw	Raw
	JL703_JL697_FtularensisLVS_Digest_2uGin1uL_0l5uL_000min_02.msf	Msf
	JL703_JL697_FtularensisLVS_Digest_2uGin1uL_0l5uL_000min_02.raw	Raw

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Publications

Sense and Nonsense of Elevated Column Temperature in Proteomic Bottom-up LC-MS Analyses.

Lenčo Juraj J Šemlej Tomáš T Khalikova Maria A MA Fabrik Ivo I Švec František F

Journal of proteome research 20201021 1

Elevated column temperature represents a simple means for improving chromatographic separation of peptides. Here, we demonstrated the advantages of the column temperature in peptide separation using state-of-the-art columns. More importantly, we also determined how temperature can impair proteomic bottom-up analyses. We found that an elevated temperature in combination with the acidic pH of the mobile phase induced in-column peptide hydrolysis with high specificity to Asp and accelerated five mo ...[more]

PMID: 33085896

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Reversed-phase liquid chromatography of peptides for bottom-up proteomics: A tutorial

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Mitigating in-column artificial modification in HTLC for bottom-up proteomics and quality control of biopharmaceuticals

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The identification of bacterial protein interaction partners pointed out to new intracellular functions of Francisella tularensis glyceraldehyde-3-phosphate dehydrogenase

Project description:Glyceraldehyde-3-phopshate dehydrogenase (GAPDH) is well-known for its involvement in numerous non-metabolic processes inside the mammalian cells. Alternative functions of prokaryotic GAPDH are mainly deduced from its extracellular localization and the ability to bind to selected host proteins. Data on its participation in intracellular bacterial processes are scarce as there is so far only one study dealing with this issue. We previously reported several evidence that the GAPDH homologue of Francisella tularensis, GapA, might also exert additional non-enzymatic functions. To follow up our former observation, here we decided to find out GapA’s interacting partners within the bacterial proteome to explore its new roles at the intracellular level. The quantitative proteomics approach based on stable isotope labelling of amino acids in cell culture (SILAC) in combination with affinity purification and mass spectrometry enabled us to identify 18 proteins potentially interacting with GapA, six of those interactions were further confirmed by alternative methods. Half of the identified proteins were involved in non-metabolic processes. Further analysis together with the quantitative label-free comparative analysis of proteomes isolated from the wild-type strain and strain with deleted gapA gene suggests that the GapA is implicated in DNA repair processes. The absence of GapA promotes secretion of its most potent interaction partner, the hypothetical protein with peptidase propeptide domain (PepSY) indicating its impact on subcellular distribution of some proteins.

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Improved reversed phase chromatography of hydrophilic peptides from spatial and temporal changes in column temperature

Project description:Reversed phase chromatography is an established method for peptide separation and frequently coupled to electrospray ionization-mass spectrometry for proteomic analysis. Column temperature is one parameter that influences peptide retention and elution, but it is often overlooked as its implementation requires additional equipment and method optimization. An apparatus that allows temperature manipulation in three areas of a two-column setup was evaluated for improvements in chromatography. Using commercially available standards, we demonstrate that a low column temperature (0 °C) during sample loading enhances the peak shape of several bovine serum albumin hydrophilic peptides. For digested HeLa lysates, approximately 15% more peptide identifications were obtained by increasing the precolumn temperature to 50 °C after the 500 ng sample was loaded at a low temperature. This method also identified additional early eluting peptides with grand average of hydropathicity values less than -2. We also investigated the effect of cooler column temperatures on peptides with post-translational modifications. It was possible to minimize the co-elution of an isoaspartylated peptide and its unmodified version when the analytical column temperature was decreased to 5 °C. Aside from demonstrating the utility of lower temperatures for improved chromatography, its application at specific locations and time points is critical for peptide detection and separation.

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Proteomic signatures of antimicrobial resistance in Yersinia pestis and Francisella tularensis

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The characterization of column heating effect in nano-flow liquid chromatography mass spectrometry (nanoLC-MS)-based proteomics

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2020-01-09 | PXD017016 |

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Expression data from mouse liver infected with Ft LVS (without or with LPS pretreatment)

Project description:Background: It has been shown previously that administration of Francisella tularensis (Ft) LVS lipopolysaccharide (LPS) protects mice against subsequent challenge with Ft LVS and blunts the pro-inflammatory cytokine response. Methods: To investigate further the molecular mechanisms that underlie Ft LVS LPS-mediated protection, we profiled global hepatic gene expression following Ft LVS LPS or saline pretreatment and subsequent Ft LVS challenge using Affymetrix arrays. Results: A large number of genes (> 3000) were differentially expressed at 48 hours post-infection. The degree of modulation of inflammatory genes by infection was clearly attenuated by LPS-pretreatment in the surviving mice. However, LPS alone had a subtle but significant effect on the gene expression profile of the uninfected mice. By employing gene set enrichment analysis, we discovered significant up-regulation of peroxisome proliferator activated receptors (PPARs) and their target genes in LPS-treated liver. Conclusions: We hypothesize that the LPS-induced blunting of pro-inflammatory response in mouse is, in part, mediated by PPARs (alpha and gamma). Experiment Overall Design: Two groups of 9 mice each were used for this experiment. 48 hours prior to Ft LVS challenge, mice were injected i.p. with either 100 ng of Ft LVS LPS or an equivalent volume of saline. On the day of challenge, 3 saline- and 3 Ft LVS LPS-pretreated animals were sacrificed (uninfected controls), while all remaining mice were challenged i.p. with ~4-5 X 105 Ft LVS. Ft LVS-challenged mice were sacrificed (in groups of 3) at 24 and 48 hours post-infection.

2010-02-21 | E-GEOD-16207 | biostudies-arrayexpress