Here are a few thoughts:

(1) the band you cut out was a truncation product. Maybe the protein is indeed cleaved in your tissue sample, or maybe a sufficient amount of protease inhibitors were not included during the lysis. LC-MS/MS would identify and sequence peptides; then a series of software are used to match the experimental peptides to theoretical digests from proteins. So unless the truncated form of the protein was also included in the search database, the software would, by default, match identified peptides to the full-length protein

(2) Proteins like actin are *very* abundant in cells - so when you load lysate on an SDS-PAGE gel, these abundant proteins often streak through the entire lane. When you use an antibody to detect your signal, you go for very short exposures, so don't see the streaking. A mass spec *will* see it. So the actin in your 250 kDa band could just be a result of overloading the gel. Good practice includes loading small amounts of protein (remember mass specs are very sensitive - I routinely load 10 microgram of total lysate in a lane and stain with Sypro Ruby, then cut bands out for LC-MS/MS) and using an excess of a strong reducing agent such as TCEP (Sigma sells it) while running your sample. I've found that TCEP gives much less streaking than DTT.

(3) Proteins often do not migrate according to their true MW on SDS-PAGE. Take a well-studied protein like p53, for example. It's MW is *not* actually 53 kDA - it was merely named p53 because it ran at 53 kDa on SDS-PAGE. 

I hope that helps - good luck!

Hi,

of course, some larger proteins can have degradation products that run much lower and others can aggregate (maybe because of overloading as suggested above) in the gel so they appear at much higher MW, especially when the differences are so dramatic, it's less likely to be caused by PTMs.

Have you considered the most simple explanation which is mixing up of samples? Don't get me wrong but these two examples could well be the result of confusing the samples when the expected size of one matches roughly (within ~10% of the expected MW) the detected MW of the other one.

Best regards

Hüseyin

Hi Eric,

All the previous answers point to the relevant reasons for the observation. Some proteins just don´t separate well and end up distributed over the whole gel. Some proteins are actually contaminants (Actin is a good candidate for such a contaminant) intruduced during the sample preparation and are also present in most slices. It would be nice to have ways to validate whether a protein is indeed a carryover due to electrophoresis, or it is a degradation product of a protein present in a higher MW gel slice. This can be somehow easily achieved using the following points:

- Perform in gel digest of the slices from whole gel lane rather than selected gel slices

- Analyze the data on a high resolution insturment (e.g. Orbitrap)

- Analyze the data using MaxQuant. I am putting this point since the output tables from this software are very suitable for data analysis.

- Normalize the protein intensity (summed XIC of all peptides from the proteins) across gel slices to the highest intensity in a gel slice.

- Follow intensity profiles

What you want is to arrive at a global view like in supplementary figure 12:

http://onlinelibrary.wiley.com/doi/10.1002/pmic.201300035/suppinfo

but for your purposes you can focus on the profiles of specific proteins. If they have a single peak over the entire gel lane, while the protein is identified in a number of slices, then the reason is, usually, carryover. When you observe more than one peak in the intensity profiles (you should consider the interplay between protein abundance and limit of detection for lowly abundant proteins) then probably there was a protein modification event, e.g. proteolysis, either biological or related to sample preparation.

Ilian

Eric,

all of the above answers are very sensible and give you excellent pointers. I would only like to add two small points:

Don't trust the SDS-PAGE gel. MWt estimates on SDS-PAGE can be close to the 'true' molecular weight of a particular protein, but they can also be way off. Aberrant migration on SDS-PAGE is a well known phenomenon (one protein that comes to my mind is MARCKS, that migrates at about 80 kDa, but has an actual molecular weight of about 28 kDa. These aberrant migrations are caused amongst others by differences in the number of SDS-molecules that can attach to a particular protein.

Secondly, don't trust the MWt information that you obtain from databases. The MWt in your search result will be a calculated theoretical MWt that is calculated from the gene sequence of the relevant entry in a genomic database. It will not include the many post-translational modiciations a protein can undergo, including proteolysis (e.g. cleavage of a signal peptide), glycosylation and lipid modifications, all of which can have a strong influence on a proteins migration (apparent MWt estimate) on SDS-PAGE.

Hope this helps,

Achim