Alkylation and Mass Spectrometry Analysis of WT Coa6

A sample of ^WTCoa6 (200 μM) was reduced with 4 mM DTT and then alkylated with 50 mM IAA for 30 min before MALDI-TOF. In addition, IAA-labeled and unlabeled samples were subjected to trypsin digestion and tandem MS/MS sequencing using a Thermo Fisher Scientific LTQ Orbitrap Elite ETD Mass Spectrometer as previously reported (56 (link)). Upon digestion, peptides containing the free sulfhydryl groups on the cysteine residues shift by 58 Da because of the alkylation. The predicted mass of alkylated peptides were calculated using PEPTIDEMASS (57 (link)).

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Maghool S., Cooray N.D., Stroud D.A., Aragão D., Ryan M.T, & Maher M.J. (2019). Structural and functional characterization of the mitochondrial complex IV assembly factor Coa6. Life Science Alliance, 2(5), e201900458.

Publication 2019

LTQ Orbitrap Elite ETD Mass Spectrometer

Alkylation Cysteine Digestion Maldi Ms ms Peptides Sulfhydryl Trypsin

Corresponding Organization : University of Melbourne

Other organizations : Australian Synchrotron, Australian Nuclear Science and Technology Organisation, Australian Regenerative Medicine Institute, Monash University

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Variable analysis

independent variables

Reduction with 4 mM DTT
Alkylation with 50 mM IAA for 30 min

dependent variables

Peptide mass shifts due to alkylation
Peptide identification and sequencing by MALDI-TOF and tandem MS/MS

control variables

Sample of ^WTCoa6 (200 μM)
Trypsin digestion of IAA-labeled and unlabeled samples

controls

Positive control: IAA-labeled samples
Negative control: Unlabeled samples

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