Proteomic Identification of Differentially Expressed Proteins

Protein spots with significant differences between the two groups were excised, dehydrated in acetonitrile, and dried at room temperature^{28 (link)}. Gel pieces were denatured, alkylated, trypsin digested and analysed by an Ultraflex II MALDI-TOF-TOF mass spectrometer (Bruker Daltonics GmbH, Bremen, Germany) under the control of FlexControl TM 2.4 software (Bruker Daltonics GmbH)^{28 (link)}. Acquired peptide mass fingerprint (PMF) were processed using the software Flex Analysis^TM 3.0 (Bruker Daltonics, Bremen, Germany)^{28 (link)}. The peak detection algorithm was: SNAP (Sort Neaten Assign and Place); S/N threshold: 1.5; Quality Factor Threshold: 50. The tryptic auto-digestion ion picks (trypsin [108–115] 842.5094 Da, trypsin [58–77] 2211.104 Da) were used as internal standards^{28 (link)}. The resulting peptide mass lists were used to search the Matrix science database (http://www.matrixscience.com)^{28 (link)}. The following search parameter criteria were used^{65 (link)}: mass tolerance 100 ppm, miss cleavage ≤ 1, modification comprises Carbamidomethyl and methionine oxidation^{28 (link)}. Matched peptides number between experimental PMF and theoretical PMF ≥ 5^{28 (link)}.

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Zhao J., Qin H., Xin J., Liu N., Han R., Perez-Campo F.M, & Li H. (2020). Discovery of genes and proteins possibly regulating mean wool fibre diameter using cDNA microarray and proteomic approaches. Scientific Reports, 10, 7726.

Publication 2020

Ultraflex II MALDI-TOF-TOF mass spectrometer

Acetonitrile Cleavage Digestion Maldi Methionine Peptide Peptide fingerprint Protein Spots Tolerance Trypsin

Corresponding Organization :

Other organizations : Qingdao Agricultural University, Universidad de Cantabria, Qingdao Academy of Agricultural Sciences

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Variable analysis

independent variables

Protein spots with significant differences between the two groups

dependent variables

Peptide mass fingerprint (PMF) of the excised and processed protein spots

control variables

Tryptic auto-digestion ion picks (trypsin [108–115] 842.5094 Da, trypsin [58–77] 2211.104 Da) used as internal standards

controls

Positive control: Tryptic auto-digestion ion picks used as internal standards
Negative control: Not explicitly mentioned

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