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My expressed proteins are frequently truncated and I'm trying to figure out which bands are which. The first thing to come to mind is using PeptideCutter from ExPASy but there is just a data deluge of potential sites. I was curious what other strategies exists for determining the potential breaks asides using LC-MS.

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Are you using E. coli for expression? Using a protease-deficient strain (eg BL21DE3) is usually a good idea for testing this stuff. If you still have truncations, then an analysis of the DNA sequence is in order. –  Thomas Ingalls Feb 24 '12 at 20:59
I am using an E. coli expression strain but not BL21. Probably worth a try. I have tried using protease inhibitors and those haven't worked. –  bobthejoe Feb 24 '12 at 21:01
I should add that I've been using cell-free protein expression. However, the question at hand is completely independent of the expression system used. –  bobthejoe Feb 28 '12 at 19:19

3 Answers 3

LC-MS is certainly quantitative and will give you a definitive answer, but it is costly and requires access to such a machine.

I presume you're analyzing your protein based on western blotting.

The first thing you should always do is verify your DNA sequence is coding for the protein product you want. Once you're sure of this, the western blot will give you an indication as to where your protein is (approximately) being cleaved. Say you protein has a predicted size of 40 kDa and you see a band at 20 kDa, then your cleavage is somewhere in the middle of your sequence. There are a TON of proteases that could be potentially cleaving your peptide and you need to have a hypothesis as to what that could be. Your peptide could be cleaved by an endopeptidase (cutting within), or a carboxypeptidase (C-terminal cleavage) or an aminopeptidase (N-termainal cleavage). To go back to the 20/40 kDa example, it could be your peptide was cleaved N-terminally o C-terminally down to a size of 20 kDa, or that it was literally cleaved in the middle by an endopeptidase.

Something you may consider is the use of a general protease inhibitor cocktail (Roche makes a really good tablet product called Complete and Complete mini tab). These mixes have a bunch of general protease inhibitors which will stop most cleavage events. If you still see cleavage after using an inhibitor cocktail, you can reasonably expect that you are not inhibiting the protease with the tablet (thus narrowing down your search) and then you can better comb through Expasy's PepCutter data. You should also do a literature search to see what has been reported about your peptide or motif.

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+1. Tried using the cOmplete tablets and still seeing problems. Do you have a good idea what's in the mix? –  bobthejoe Feb 28 '12 at 2:59
From the manufacturer: roche-applied-science.com/PROD_INF/BIOCHEMI/No1_99/pgs21-22.pdf Notice that inhibition activity is not maximal, and decreases with time. Collection and manipulation of your samples must be done chilled and as quickly as possible to avoid degradation. –  leonardo Feb 28 '12 at 16:03

You could also try induction at lower temperatures to minimize chances of protein degradation. Cells such as Arctic cells (http://tinyurl.com/arcticecoli) are optimized for low temperature expression although BL21 works just as good for the proteins we express (upto 140kDa in size)

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Good answer that is relevant to the problem but not relevant to the question. –  bobthejoe Feb 27 '12 at 6:07
I have tried lower temperature expression at 25C but still see issues. I could go lower to 10C. –  bobthejoe Feb 28 '12 at 1:32
@bobthejoe 10C is probably too low - try 16C first. –  Amy Feb 28 '12 at 17:38

One way to learn more about how your protein is being processed/degraded is to add two different tags on either end of your protein. Express your protein, then blot with the two antibodies for your tags. The most likely scenario here is that one end or another is being cleaved and then degraded - you'll be able to tell which end by what tag epitope remains. And since you'll know the approximate size, you can narrow down the region where the processing occurs.

Small tags such as HA, cMyc, Flag or 6xHis work well because they are less likely to be cleaved off themselves.

The only way to know for certain how and where your protein is being truncated is empirically: either by N-terminal sequencing of the fragment(s) by mass spec or by mutating the putative processing sites and testing the expression again.

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An epitope tag can tell you from a western blot where the protein is being cleaved (N-terminally, C-terminally, somewhere in the middle) without antibodies raised specifically for localized epitopes. Mutation of putative cleavage sites is a more specific way to go if you can identify what is cleaving the protein. –  leonardo Mar 16 '12 at 1:46

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