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Using instrument logic to improve identifications in low stoichiometry SILAC/TMT experiments

ABSTRACT: Pulsed SILAC approaches allow measurement of protein dynamics, including protein translation and degradation. However, its use in quantifying acute changes has been limited due the low labeled peptide stoichiometry. Here, we describe the use of instrument logic to select peaks of interest via targeted mass differences (TMD) for overcoming this limitation. Comparing peptides artificially mixed at low heavy-to-light stoichiometry measured using standard data dependent acquisition with or without TMD revealed 2-3 fold increases in identification without significant loss in quantification precision for both MS2 and MS3 methods. Our benchmarked method approach increases throughput by reducing the necessary machine time. We anticipate that all pulsed SILAC measurements, if combined with TMT or not, would greatly benefit from instrument logic based approaches.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Uterine Cervix

SUBMITTER: Kevin Klann

LAB HEAD: Christian Münch

PROVIDER: PXD018445 | Pride | 2020-05-29

REPOSITORIES: Pride

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			Action	DRS
	125ms.msf	Msf
	180ms.msf	Msf
	20191224_DDA_20191228094911.raw	Raw
	20191224_TMD_both_20191228123611.raw	Raw
	20191230_DDA_MS3.raw	Raw

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Publications

Instrument Logic Increases Identifications during Mutliplexed Translatome Measurements.

Klann Kevin K Münch Christian C

Analytical chemistry 20200529 12

Pulsed Stable Isotope Labeling in Cell culture (SILAC) approaches allow measurement of protein dynamics, including protein translation and degradation. However, its use for quantifying acute changes has been limited due to low labeled peptide stoichiometry. Here, we describe the use of instrument logic to select peaks of interest via targeted mass differences (TMD) for overcoming this limitation. Comparing peptides artificially mixed at low heavy-to-light stoichiometry measured using standard da ...[more]

PMID: 32442378

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