Abstract
BackgroundOptimal protocols for efficient and reproducible protein extraction from formalin-fixed paraffin-embedded (FFPE) tissues are not yet standardised and new techniques are continually developed and improved. The effect of polyethylene glycol (PEG) 20 000 on protein extraction efficiency has not been evaluated using human FFPE colorectal cancer tissues and there is no consensus on the protein extraction solution required for efficient, reproducible extraction.ObjectiveThe impact of PEG 20 000 on protein extraction efficiency, reproducibility and protein selection bias was evaluated using FFPE colonic tissue via liquid chromatography tandem mass spectrometry analysis.MethodsThis study was conducted from August 2017 to July 2019 using human FFPE colorectal carcinoma tissues from the Anatomical Pathology department at Tygerberg Hospital in South Africa. Samples were analysed via label-free liquid chromatography tandem mass spectrometry to determine the impact of using PEG 20 000 in the protein extraction solution. Data were assessed regarding peptide and protein identifications, method efficiency, reproducibility, protein characteristics and organisation relating to gene ontology categories.ResultsPolyethylene glycol 20 000 exclusion increased peptides and proteins identifications and the method was more reproducible compared to the samples processed with PEG 20 000. However, no differences were observed with regard to protein selection bias. We found that higher protein concentrations (> 10 µg) compromised the function of PEG.ConclusionThis study indicates that protocols generating high protein yields from human FFPE tissues would benefit from the exclusion of PEG 20 000 in the protein extraction solution.
Highlights
1: Precursor Tolerance Unit: ppm 2Precursor Ion m/z Tolerance: 10.0 3: Fragment Ion Tolerance Unit: Da 4: Fragment Ion m/z Tolerance: 0.02 5: Cleavage: Enzyme 6: Enzyme: Trypsin 7: Missed Cleavages: 2 8: Specificity: Specific 9: Database: uniprot-human-ref-proteome-trypsin-Oct2018_concatenated_target_decoy.fasta 10: Forward Ion: b 11: Rewind Ion: y 12: Fixed Modifications: Methylthio of C 13: Variable Modifications: Oxidation of M, Deamidation of N, Deamidation of Q 14: Refinement Variable Modifications: Acetylation of protein N-term, Pyrolidone from E, Pyrolidone from Q, Pyrolidone from carbamidomethylated C 15: Refinement Fixed Modifications: Methylthio of C
4: ProteinsFNR Limit [%]: 18.75 5: Proteins: Confidence Limit [%]: 77.32 6: Proteins: PEP Limit [%]: 22.68 7: Proteins: Confidence Accuracy [%]: 0.13 8: Peptides (Unmodified): #Validated: 8061.0 9: Peptides (Other): #Validated: 919.0 10: Peptides (Unmodified): Total Possible TP: 8925.99 11: Peptides (Other): Total Possible TP: 1604.24 12: Peptides (Unmodified): FDR Limit [%]: 0.98 13: Peptides (Other): FDR Limit [%]: 0.87 14: Peptides (Unmodified): FNR Limit [%]: 10.75 15: Peptides (Other): FNR Limit [%]: 43.25 16: Peptides (Unmodified): Confidence Limit [%]: 79.63 17: Peptides (Other): Confidence Limit [%]: 91.62 18: Peptides (Unmodified): PEP Limit [%]: 20.37 19: Peptides (Other): PEP Limit [%]: 8.38 20: Peptides (Unmodified): Confidence Accuracy [%]: 0.1 21: Peptides (Other): Confidence Accuracy [%]: 0.53 22: PSMs (Charge 2): #Validated PSM: 6715.0 23: PSMs (Charge 3): #Validated PSM: 2897.0 24: PSMs (Charge 4 and Charge 5, 6): #Validated PSM: 364.0 25: PSMs (Charge 2): Total Possible TP: 8150.28 26: PSMs (Charge 3): Total Possible TP: 3445.06 27: PSMs (Charge 4 and Charge 5, 6): Total Possible TP: 393.55 28: PSMs (Charge 2): FDR Limit [%]: 1.0 29: PSMs (Charge 3): FDR Limit [%]: 0.97 30: PSMs (Charge 4 and Charge 5, 6): FDR Limit [%]: 0.82 31: PSMs (Charge 2): FNR Limit [%]: 18.53 32: PSMs (Charge 3): FNR Limit [%]: 16.89 33: PSMs (Charge 4 and Charge 5, 6): FNR Limit [%]: 8.42 34: PSMs (Charge 2): Confidence Limit [%]: 78.64 35: PSMs (Charge 3): Confidence Limit [%]: 79.11 36: PSMs (Charge 4 and Charge 5, 6): Confidence Limit [%]: 83.33 37: PSMs (Charge 2): PEP Limit [%]: 21.36 38: PSMs (Charge 3): PEP Limit [%]: 20.89 39: PSMs (Charge 4 and Charge 5, 6): PEP Limit [%]: 16.67 40: PSMs (Charge 2): Confidence Accuracy [%]: 0.19 41: PSMs (Charge 3): Confidence Accuracy [%]: 0.45 42: PSMs (Charge 4 and Charge 5, 6): Confidence Accuracy [%]: 9.09
1: PeptideShaker Version1.16.31 2: Date: Sat Dec 15 11:10:38 CAT 2018 3: Experiment: 1068_Pellet_noPEG_HILIC_2_PEG-pilot-study 4: Sample: 1068_Pellet_noPEG_HILIC_2_PEG-pilot-study 5: Replicate Number: 0 6: Identification Algorithms: X!Tandem, MS Amanda and MS-GF+.
Summary
Precursor Ion m/z Tolerance: 10.0 3: Fragment Ion Tolerance Unit: Da 4: Fragment Ion m/z Tolerance: 0.02 5: Cleavage: Enzyme 6: Enzyme: Trypsin 7: Missed Cleavages: 2 8: Specificity: Specific 9: Database: uniprot-human-ref-proteome-trypsin-Oct2018_concatenated_target_decoy.fasta 10: Forward Ion: b 11: Rewind Ion: y
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