FASP (Filter-Aided Sample Prep)
In-solution Tryptic Digestion
In-gel Tryptic Digestion
SDS-PAGE protein separation
SILAC media preparation
Basic Guidelines for Protein
Sample Handling and Preparation
preparation of a protein sample (or protein complex) is the key to successful
mass spectrometry. It is very important to prevent or minimize contamination of
your sample. Problem contaminants include keratins, polyethyleneglycol (PEG) and detergents.
is a common protein contamination. It is extremely easy to contaminate your
samples with keratins, which are ubiquitous and come from skin cells, hair,
fingerprints, lint, lint from Kim-wipes, and dust in the lab. The
smallest speck of dust or lint will most likely overwhelm your sample with keratin
proteins. Great care is required to prevent keratins from showing up in MS
the amount of exposure of the gel, gel equipment, and supplies to primary
sources of keratins, such as skin, hair, clothing, etc. Reduce the amount of
exposure to dust and particulates, both of which can be a rich source of
b. Perform as much work as possible
in a biological safety cabinet (BSC) or laminar flow hood.
c. Thoroughly wipe down work
surfaces with water and ethanol.
powder-free nitrile gloves. Don’t use Latex gloves. Latex is manufactured with
proteins, which can contaminate your protein samples.
“low-binding” (but not silanized) polypropylene microcentrifuge tubes is
recommended for sample storage and to prevent adsorptive losses of proteins and peptides.
f. Use precast gels such as NuPage
gels from Invitrogen and use their readymade buffers, loading dye, etc to
minimize keratin contamination.
g. Gel tanks are a common source of
contaminants. If your gel tank has been left to dry by the side of the sink for
several days it is now contaminated with keratin from dust.
h. When staining your gel, cover the staining tray on the shaker and
wash the staining dish well before use.
i. Communal lab chemicals (loading
buffer, lab buffers, etc) are the most common sources of keratin. Do not use –
PEG is strongly ionized and can drown out signal from the analyte of interest. PEG has a common repeat of 44 Da
in the mass spectrum.
a. Avoid dish soap as it contains PEG. Either do not use or rinse glassware with very hot
water followed by an organic solvent rinse.
HPLC grade solvents.
c. Do not
store organic solvents in plastic tubes. PEG contaminants can leach out of
recommend Eppendorf brand microcentrifuge tubes.
For in-gel submissions, most of the
detergents (<0.1% SDS, etc) can be cleared from the system before mass
spectrometry analysis. Avoid using Triton-X100, SDSAPS, Tween 20, NP-40, etc, which can adversely affect results. If you need to clean up your sample, you can
use detergent spin columns, gel filtration, dialysis, etc. to remove the
excess detergents or use the Filter Aided Sample Preparation (FASP) method for sample preparation. Please
utilize one of the mass spec friendly detergents listed below:
Mass Spec Friendly Detergents:
Acid Labile Surfactants