Selected reaction monitoring based analysis of proteins in liver tissue of Labeo rohita
Nissa MU, Pinto N, Varshnay A, Goswami M, Srivastava S. Ecological Monitoring and Omics: A Comprehensive Comparison of Workflows for Mass Spectrometry-Based Quantitative Proteomics of Fish (
Labeo rohita) Liver Tissue. OMICS. 2022 Sep;26(9):489-503. doi: 10.1089/omi.2022.0086. Epub 2022 Aug 26. PMID: 36036978.
- Organism: Labeo rohita
- Instrument: TSQ Altis
- SpikeIn:
Yes
- Keywords:
FASP, method optimization, rohu liver, SRM, STRAP
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Lab head: Sanjeeva Srivastava
Submitter: Sanjeeva Srivastava
Bottom-up proteomics analysis relies on efficient protein digestion for performing mass spectrometric analysis. Optimisation of protein solubilisation and digestion strategies is a key step for obtaining reliable proteomic data. In this study, two different solubilisation buffers (SDS and Urea) and three digestion methods (FASP, S-Trap and in-solution,) were applied for the proteomic analysis of liver tissue of Labeo rohita. Label free quantification analysis was performed to analyse the similarities and differences in the results obtained in each method. Based on the discovery data, few proteins were selected for targeted data acquisition. These proteins were chosen on the basis of highest peptide spectral matches in different experimental conditions. The aim was to validate the trends in abundance of proteins in different experimental conditions.
The experiment was conducted using an HPLC-Dionex Ultimate 3000 system (Thermo Fisher Scientific, USA) linked to a TSQ Altis Mass Spectrometer (Thermo Fisher Scientific, USA). The peptides were separated for 10 minutes using a Hypersil Gold C18 column at a flow rate of 0.45 µL/min. The binary buffer system used 0.1 percent Formin acid (FA) as buffer A and 80 percent Acetonitrile as buffer B in 0.1 percent FA.
Transition lists were generated by importing the list of selected proteins into Skyline software (version 21.1.0.146). The initial lists of peptides were run in pool samples which were further refined and two lists covering 713 transitions corresponding to 106 peptides of five proteins were run across all the samples. Seven transitions for a synthetic peptide AVLTIDEK (labelled at C-terminal Lysine) were also included for the final run and all samples were spiked in with equal amount of the synthetic peptide for monitoring the consistency of mass spectrometric runs. In order to monitor the day-wise instrument response, 500 ng of a human cell line sample (for peptides of two proteins- ALDOA and GAPDH) was run during the experiment.
Two skyline documents have been shared in this experiment:
1. QC list of peptides (100 transitions, 9 peptides) monitored in a human cell line sample to check the instrument response throughout the experiment.
2. Target peptide list run across the samples (Refined 642 transitions, 98 peptides including a heavy labelled synthetic peptide),
The SRM data was acquired for a total of 12 samples belonging to four conditions, SF, SS, UF and UIS based on the experiment. Each of this condition consisted of 3 replicates (two individual samples and their pool e.g., SF1, SF2 and SF-pool, SS1, SS2, SS-pool respectively and so on). The data was acquired for two transition lists for all the samples resulting in 24 raw files.
After data acquisition, condition wise analysis was performed in Skyline.
Created on 10/7/21, 8:03 PM