What are the methods for PTMScan® mass spectrometry?

The methods for the LC and MS are shown below:

	Parameter	Setting
*Sample prep*	IP sample resuspension volume	20 uL
	Sample volume per injection	4 uL
	Resuspension buffer	5% acetonitrile/0.1% trifluoroacetic acid

*Chromatography*	Column dimensions	100 um ID x 45 cm long
	Stationary phase	Sepax GP C18, 1.8um, 120A
	Solvent A	0.125% formic acid in water
	Solvent B	0.125% formic acid in acetonitrile
	Flow rate	275 nL/min
	Gradient	0 min - 3% B, 1 min - 6% B, 91 min - 38% B, 141 min - 100% B, 142 min - 3%B, 154 min (end) - 3% B

*Mass Spectrometry*	Instrument model	Thermo Fusion Lumos
	MS1 detector / resolving power	Orbitrap / 120000
	MS1 scan range	300-1500 m/z
	DDA mode	3s cycle time
	Isolation width	1.6 m/z
	Fragmentation mode	HCD
	MS2 detector / resolving power	Orbitrap / 60000

*Database Search*	Search Platform	Comet
	Database	Mouse SwissProt
	Precursor match tolerance	(+/-)50 ppm
	Product ion match tolerance	(+/-) 0.02 Da
	Post-search filter on precursor mass	(+/-) 5 ppm
	FDR cutoff	5%

These parameters can also be found in the “Methods Overview” tab on the example data Excel files.

Last updated: March 6, 2023

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