N-terminal sequencing/iTRAQ

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samm
samm's picture
N-terminal sequencing/iTRAQ

Can small cleavage proteins (~15kDa) be subject to N-terminal sequencing by mass spec? Is a deglycosylation step necessary? Is it better to use multiple "digest" enzymes?
Also, is N-terminal labelling using reagents such as iTRAQ helpful in sequencing and comparision?

NunoBandeira
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samm wrote:Can small cleavage

samm wrote:

Can small cleavage proteins (~15kDa) be subject to N-terminal sequencing by mass spec? Is a deglycosylation step necessary? Is it better to use multiple "digest" enzymes?
Also, is N-terminal labelling using reagents such as iTRAQ helpful in sequencing and comparision?

Dear Samm,

Proteins this small can very likely be sequenced even without deglycosylation or N-terminal labelling. If it would be easy for you to separately digest your sample with different enzymes and analyse it using Tandem Mass Spectrometry then our Shotgun Protein Sequencing approach should be able to accurately reconstruct your protein. Below is the reference to the paper where we described our methodology.

Best regards,

Nuno Bandeira

Shotgun Protein Sequencing by Tandem Mass Spectra Assembly.
Anal Chem. 2004 Dec 15;76(24):7221-33.
Bandeira N, Tang H, Bafna V, Pevzner P.

The analysis of mass spectrometry data is still largely based on identification of single MS/MS spectra and does not attempt to make use of the extra information available in multiple MS/MS spectra from partially or completely overlapping peptides. Analysis of MS/MS spectra from multiple overlapping peptides opens up the possibility of assembling MS/MS spectra into entire proteins, similarly to the assembly of overlapping DNA reads into entire genomes. In this paper, we present for the first time a way to detect, score, and interpret overlaps between uninterpreted MS/MS spectra in an attempt to sequence entire proteins rather than individual peptides. We show that this approach not only extends the length of reconstructed amino acid sequences but also dramatically improves the quality of de novo peptide sequencing, even for low mass accuracy MS/MS data.

samm
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Thanks very much! I'll look

Thanks very much! I'll look this up. One more query - how are the results affected if there are multiple proteins - I'm trying to use IPs/aff purif, but suspect that there could be more than one cleavage product at similar sizes.

Roshan
Roshan's picture
samm wrote:Can small cleavage

samm wrote:

Can small cleavage proteins (~15kDa) be subject to N-terminal sequencing by mass spec? Is a deglycosylation step necessary? Is it better to use multiple "digest" enzymes?
Also, is N-terminal labelling using reagents such as iTRAQ helpful in sequencing and comparision?

Check the site:http://mcb.harvard.edu/microchem/