How does MaxEnt3 processing work for peptide peaks containing Br atom in UNIFI? - WKB23262

ENVIRONMENT

• UNIFI 1.8.2

ANSWER

Maxent3 looks for multiply charged ions and then converts them into singly charged neutral masses. In a peptide containing bromine (Br), there will be overlapping carbon isotope series (one for 79Br and another for 81Br), which makes things more complicated. Normally, the first isotope in a series is the C12 peak, and Maxent3 can look for that as the start of the ion series and do the deconvolution calculation from that. In a peptide containing bromine, the 81Br/12C peak and the 79Br/14C peak have the same mass, for example:

440.62  = 79Br/12C
441.87 = 79Br/13C
441.12 = 79Br/14C, and also 81Br/12C
441.3722 = 81Br/13C
441.622 = 81Br/14C

This means the third peak in the group (the start of 81Br isotope series) may be the most intense.  

Because Maxent bases deconvolution on the most intense peak being the 12C (monoisotopic) peak, it may mistakenly use just the 81Br ion series, starting with 441.1896 (4+) and 587.9114 (3+).

It is possible to match the observed deconvoluted mass to the expected mass of the target component, by incorporating the extra 2Da from the 81Br into the expected mass of the peptide sequence. If the bromine is added to a side chain of an existing amino acid, you can incorporate it into the search method by defining an amino acid modified with the formula “81Br”, and then selecting the amino acid to which the modification applies. If the peptide containing Br is a non-standard amino acid incorporated into the peptide, you should define a custom amino acid in the scientific library and use [81Br] in the formula to specify the heavy bromine isotope, for example C6H14N2O2[81Br].

Alternatively, in some cases it may be possible to get UNIFI to recognize both the 79Br and the 81BR peaks as separate components. To do so, set up the 81Br labeled synthetic amino acid within the amino acids library, and then create a second target sequence containing the 81Br version of the custom amino acid. Then, when you set up the processing parameters, define target sequences (the 79Br and 81Br versions) and, in the 3D Peak Detection settings, set Chromatographic peak width to Manually specified peak width = 0.03 minutes.

ADDITIONAL INFORMATION