Mass Spectrometry Protocols

Our protocols for mass spectrometry have been developed to provide maximum recovery of peptides post-digestion and to ensure that interfering agents (e.g. SDS, urea, etc.) are not present. Robotic digestion also minimizes the risk of keratin contamination and allows for high throughput sample processing. Specific for MALDI analysis a preparation of matrix (a-cyano 4-hydroxy-cinnamic acid) has been developed to provide a homogeneous crystal size and maximum signal response across the entire spot area, thus minimizing the reliance on “hot spots”. We also utilize µC18 ZipTips to eliminate salts that cause a rising baseline at low m/z.

In-Gel Digestion Protocol

The protocol we follow for in-gel digestion (including reduction and alkylation) is as follows:

Reagents

25 mM ammonium bicarbonate (ABC)
10 mM dithiothreitol (DTT) in H2O
100 mM iodoacetamide (IA) in H2O
20 µg lyophilized modified trypsin (Promega)
(The trypsin is dissolved in 200 µL of 0.01% formic acid (aq) immediately prior to initiation of the digestion protocol)
3% formic acid (aq)
Acetonitrile (ACN)

Protocol

Add 50 µL ABC to gel plugs and hold at room temperature (RT) for 10 min.
Purge liquid, add 50 µL ACN and hold at RT for 10 min.
Repeat steps 1 and 2.
Add 30 µL DTT, heat samples to 60ºC and incubate for 10 min.
Allow samples to cool for 20 min.
Purge liquid and add 30 µL IA, incubate the samples for 45 min at RT.
Purge liquid and repeat steps 1 and 2 twice.
Add 10 µL of the trypsin solution to rehydrate the gel plugs, leave for 10 min
(Note: The ProGest makes up the solution immediately prior to this step by the addition of 1.8 mL of ABC to the 200 µL trypsin solution in 0.01% formic acid to give a concentration of 10 ng/µL for digestion.)
Add 15 µL ABC and incubate the samples for 4 hr at 37ºC.
Add 7 µL 3% formic acid to stop the activity of trypsin.

The final supernatant has a volume of ~20 µL. This solution is used directly for mass spectrometric analysis. We do not use (or recommend) organic solvent (typically acetonitrile) for further extraction of peptides. See acetonitrile extraction of peptides technical note.

MALDI/MS Data acquisition is performed on an Applied Biosystems Super-DE-STR instrument. We search databases using ProFound or MS-Fit. A set of typical data is presented in our sensitivity technical note.