Biology Reference
In-Depth Information
will compete during analysis with peptides from other proteins.
Surprisingly, separation of adenovirus sample on 1D SDS-
PAGE, excising bands, in-gel digestion, and pooling of all pro-
tein bands except for the hexon was not benefi cial compared to
direct LC-MS analysis of the whole virion sample digested in-
solution. We believe, that the high capacity of the LC-MS sys-
tem in comparison to losses in the in-gel digestion approach
makes the in-solution digestion superior. However, if analyz-
ing the protein bands one by one, higher sequence coverage
for each protein is expected. Further, the sequence coverage of
a protein depends on the amino acid sequence of the proteins
in relation to what protease that is used for digesting the pro-
tein into peptides. For example, the pV and pVII proteins are
very rich in arginine and lysine residues, which are the cleavage
sites for trypsin. Then, very small peptides that cannot be
detected with the MS instrument are expected, which can
explain the low sequence coverage of those proteins. On the
other hand, proteins containing few arginines and lysines will
produce long peptides that might escape detection. In the
same way, the chances of detecting PTMs will be infl uenced by
the choice of protease.
3. In order to remove the detergents offl ine or online-coupled
trap columns can be used, for example, SCX (for anionic deter-
gents like SDS) or SAX (for cationic detergents). The sample is
loaded on such trap column and detergents remain in it, while
peptides pass through for further analysis. Unfortunately, many
peptides are also trapped by such columns, which cause sample
loss. Use of “MS-friendly” nonionic detergents might be ben-
efi cial in this case.
-octylglucoside (OBG) and RapiGest are
the best choices. OBG has signifi cantly lower impact on
LC-MS runs, and can be easily removed by ethylacetate extrac-
tion [
37
]. RapiGest is destroyed by adding 20 % TFA followed
by centrifugation, and its degradation products do not affect
LC-MS runs [
38
].
4. Quality check of sample before LC-MS: An easy way to fi nd
out if there are contaminants that infl uence the results in ESI-
LC-MS in the sample, e.g., detergents, is to use MALDI-TOF
MS analysis. Follow a general protocol for sample preparation
and analysis [
39
] and load about 0.5
β
L of your sample.
5. Control of in-solution digestion effi ciency. If having low recov-
ery of peptides after digestion it is wise to check if the digestion
has been complete. Follow the description for 1D SDS-PAGE
and load a small aliquot of sample before and after digestion.
Fix and stain the gel with Coomassie. Check that there are no
uncleaved bands in the lane of the digested sample.
6. For 2D SDS-PAGE we have tried different loading techniques
and pH intervals and found that the rehydration loading
μ
