Hi, 

I think it depends to who you ask the question.

I am to understand that you have a cloned protein (therefore you known the theoretical sequence). You performed digestion and you got a good score based on MS/MS analysis for the peptide containing the N-termini, right?

If your MS/MS spectrum shows b and y ions (at least) covering all the sequence with a very good mass error (e.g. < 50 ppm), then you can claim you have the N-termini structure.

If your MS/MS data is compatible with more than one structure (with the same strict criteria), then you cannot do the claim.

If you use MASCOT, you should have scores for more than 1 peptide. If those scores are close to each other, then it is hard to know which exact peptide you have.

All in all, it really depends on the quality of your MS/MS data. And, from my point of view, on the analytical replicates you performed (1 is not a "rule").

Dear Fabrizio,

Thank you very much of your insights :)

Hi,

You could also try to do in-source fragmentation and do top down sequencing. However, you should have a purified protein. I know that Bruker has an expertise in this. As a matrix, you have to use a "hot" matrix and more laser power than usual. I have never done it, but if you have a pure protein it should work nicely. Look here for some further information:

http://www.bruker.com/fileadmin/user_upload/8-PDF-Docs/Separations_MassSpectrometry/Literature/literature/ApplicationNotes/MT-102-Edmass-eBook.pdf

http://www.absciex.com/Documents/Downloads/Literature/mass-spectrometry-TopDown-SorceDecay-0680310.pdf

You will also find publications on pubmed.

As Fabrizio already pointed out, you can do a tryptic digestion and do MALDI ToF ToF analysis. This is also sufficient. Keep in mind that post-translational modifications occur quite frequently at the protein N-term. You should consider this as variable modifications, when you perform the database search with Mascot or another search engine. Acetylation of the protein N-term is frequently observed for proteins from eukaryotes (i.e. when you use HEK cells). 

If the N-terminal peptide is too long or too short after tryptic digestion, you can also go for other proteases such as LysC, AspN or GluC.

BestStephan

In addition to N-acetylation brought up by Stephan, we have observed oxidation of methionine and tryptophan, and cyclization of newly exposed glutamate at N-terminus, We've also observed a loss of methane sulfenic acid (CH3SOH, 64 Da) from the oxidized methionine as a major peak.  Sequencing by carboxypeptidase Y coupled with MALDI-TOF helps solving these issues (JBC 272,22758, 1997). 

Hi Stephen and Ke-Wei,

Appreciate your suggestions and insights! Will definitely read into those materials.

Have a nice weekend!

May be you can provided your are sure of the protein that is over expressed and secondly you need to have the right Matrix for it.

Thanks Jai :)

Hi. If you see in PMF the expected peptides of puryfied expressed protein and MS/MS of one of these peptides yields fragment ions matching expected fragments of N-terminal peptide then you did confirm N-terminus of your protein. Usually you can identify such a peptide by MASCOT ion-search by setting "enzyme" as "semi-trypsin" or "none". Use NCBInr database not Swiss-Prot. If your N-terminal peptide happens to have some residues from fusion protein which are not present in "natural" sequence you may not identify this peptide by database search. N-terminal sequencing (Edman) is very useful for such analysis (of course if available) because it is "de-novo" method and you may analyze intact protein from solution and after SDS-PAGE and blotting. It is not unusual to see "ragged" N-terminal sequence because of proteolysis during protein production and purification. In such case you may detect several N-terminal peptides by MS and Edman sequencing.

A tandem MS spectra from the N-terminal peptide should be sufficient to claim that this is the N-termini assuming the data was searched against a sufficiently large database and FDR analysis was performed (and reported on the PSMs. Intact mass measurement of your protein might provide additional confidence that this is the N-termini as the measured mass and calculated mass should match. This assumes that the protein is not modified or degraded or the modifications are known.