Dataset Information

Improved identification of proteoforms using FAIMS with internal CV stepping in top-down proteomics

ABSTRACT: In top-down (TD) proteomics, fractionation prior to mass spectrometric (MS) analysis is a crucial step for the high confidence identification of proteoforms and increased sample depth. In addition to liquid phase separations, gas-phase fractionation strategies such as field asymmetric ion mobility spectrometry (FAIMS) have been shown to be highly beneficial in TD proteomics. However, the need for multiple injections using different compensation voltages (CV) leads to a huge increase in measurement time and the amount of sample required. Therefore, we here investigated for the first time the use of internal CV stepping for single shot TD analysis, i.e., the application of multiple CVs per acquisition. In addition, MS parameters were optimized for the individual CVs since different CVs target certain mass ranges. For example, small proteoforms identified mainly with lower CVs can be identified with a lower resolution and number of microscans than larger proteins identified mainly with higher CVs. We investigated the optimal combination and number of CVs for different gradient lengths and validated the optimized settings with the low molecular weight proteome of CaCo 2 cells obtained by different sample preparation techniques. Compared to measurements without FAIMS, both the number of identified protein groups (+60-94%) and proteoforms (+46-127%), and their confidence were significantly increased, while the measurement time remained identical. In total, we identified 684 protein groups and 2,675 proteoforms from CaCo-2 cells in less than 24 hours using the optimized multi-CV method.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture, Colon

DISEASE(S): Colon Cancer

SUBMITTER: Andreas Tholey

LAB HEAD: Andreas Tholey

PROVIDER: PXD029792 | Pride | 2022-02-28

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	20210616_CaCo_C18MinMan_1_CID25_2h_1_FAIMS_10.raw	Raw
	20210616_CaCo_C18MinMan_1_CID25_2h_2_FAIMS_-10.raw	Raw
	20210616_CaCo_C18MinMan_1_CID25_2h_2_FAIMS_-10_20210714203748.raw	Raw
	20210616_CaCo_C18MinMan_1_CID25_2h_2_FAIMS_-10_20210715193056.raw	Raw
	20210616_CaCo_C18MinMan_1_CID25_2h_2_FAIMS_-10_allTriplicates.pdResult	Other

Items per page:

1 - 5 of 164

Similar Datasets

Project description:Episodes of chronic stress can result in psychic disorders like post-traumatic stress disorder, but also promote the development of metabolic syndrome and type 2 diabetes. We hypothesize that muscle, as main regulator of whole-body energy expenditure, is a central target of acute and adaptive molecular effects of stress in this context. Here, we investigate the immediate effect of a stress period on energy metabolism in Musculus gastrocnemius in our established C57BL/6 chronic variable stress (Cvs) mouse model. Cvs decreased lean body mass despite increased energy intake, reduced circadian energy expenditure (EE), and substrate utilization. Cvs altered the proteome of metabolic components but not of the oxidative phosphorylation system (OXPHOS), or other mitochondrial structural components. Functionally, Cvs impaired the electron transport chain (ETC) capacity of complex I and complex II, and reduces respiratory capacity of the ETC from complex I to ATP synthase. Complex I-OXPHOS correlated to diurnal EE and complex II-maximal uncoupled respiration correlated to diurnal and reduced nocturnal EE. Bioenergetics assessment revealed higher optimal thermodynamic efficiencies (ƞ-opt) of mitochondria via complex II after Cvs. Interestingly, transcriptome and methylome were unaffected by Cvs, thus excluding major contributions to supposed metabolic adaptation processes. In summary, the preclinical Cvs model shows that metabolic pressure by Cvs is initially compensated by adaptation of mitochondria function associated with high thermodynamic efficiency and decreased EE to manage the energy balance. This counter-regulation of mitochondrial complex II may be the driving force to longitudinal metabolic changes of muscle physiological adaptation as the basis of stress memory.Chronic stress targets mitochondrial respiratory efficiency in the skeletal muscle of C57BL/6 mice.Nikolic A, Fahlbusch P, Wahlers N, Riffelmann NK, Jacob S, Hartwig S, Kettel U, Dille M, Al-Hasani H, Kotzka J, Knebel B. Cell Mol Life Sci. 2023 Mar 29;80(4):108. doi: 10.1007/s00018-023-04761-4.PMID: 36988756

Dataset Information

Improved identification of proteoforms using FAIMS with internal CV stepping in top-down proteomics

Dataset's files

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets