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LC-MS/MS sample preparation

JV Jonathan G Van Vranken
SG Steven P Gygi

Last updated date: Sep 26, 2022 Views: 606 Forks: 0

original An abbreviated version of this protocol was published in eLife in Dec, 2021
Assessing target engagement using proteome-wide solvent shift assays
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We use a ZOBRAX 300Extend-C18 column (Rapid Resolution 2.1 x 250mm 3.3 micron) attached to an Agilent 1260 Infinity HPLC. Buffer A is 10 mM ammonium bicarbonate pH 8 with 5% acetonitrile. Buffer B is 10 mM ammonium bicarbonate with 90% acetonitrile. Our flow rate is 150 uL/min with the column heated to 45 C. 

 

In total, we collect 96 fractions in a deep well 96 well plate. 

 

The 96 fractions are then pooled into 24 fractions as follows: 

Fraction A1: A1, C1, E1, G1

Fraction B1: B1, D1, F1, H1

Fraction A2: A2, C2, E2, G2

Fraction B2: B2, D2, F2, H2

Fraction A3: A3, C3, E3, G3

Fraction B3: B3, D3, E3, G3

etc...

 

All 24 fractions are dried in a speed vac. 

 

Half of the fractions (either the odds [A1, B1, etc.] or the evens [A2, B2, etc.]) are stage-tipped and analyzed by LC-MS/MS. 

 

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How to cite：
Readers should cite both the Bio-protocol preprint and the original research article where this protocol was used:
  1. Van Vranken, J and Gygi, S(2022). LC-MS/MS sample preparation. Bio-protocol Preprint. bio-protocol.org/prep1965.
  2. Van Vranken, J. G., Li, J., Mitchell, D. C., Navarrete-Perea, J. and Gygi, S. P.(2021). Assessing target engagement using proteome-wide solvent shift assays. eLife. DOI: 10.7554/eLife.70784

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