Your insulin calculations are mostly correct. All you need to do now is oxidize
the cysteines by adding 3 oxygen atoms to each.
With regards to cytochrome-c, Sigma's mass value is quoted for the 'acid
modified' protein. Does anyone know the nature of this modification and how it
results in a delta mass of 24? I'll venture a guess that your first mass
calculation did not add the N-term acetyl and that you added two iron atoms to
the structure. Hence you are in error by +Fe -Acetyl. Bear in mind that heme
groups already include iron and that a single c-type heme is covalently attached
to both C's in CxxC sequences within proteins (delta mass for this
modification: 616.1772 mono; 616.5 ave.) Your last calculation has one too many
heme groups and the extra iron atoms should also not be added.
I like to use the program PAWS or GPMAW for calculating small protein and
peptide masses. These are very well thought out programs. Results from PAWS on
cytochrome-c + acetyl + heme are 12360.0 ave, and 12352.350 mono. These
calculations agree well with some programs that I've written (12360.1 ave;
12352.323mono) and with Isopro 3.0 (12360.1 ave, 12352.322 mono).
Now to digress and take the opportunity to pose my own questions concerning mass
calculations of proteins:
I consider the most reliable calculations are those where one calculates
monoisotopic mass from the atomic compostion of the molecule in question since
atomic compositions use integer values and the monoisotopic masses are known to
very high precision. Less reliable are average mass calculations on
macromolecules where it is difficult to account for natural variations in
isotopic enrichments. For average mass calculations, I believe that most of us
(in one way or another) use atomic weights derived from the average isotope
abundances determined for some comprehensive sampling of the earths crust.
These data are available from various sources (eg. CRC Handbook of Chemistry and
Physics). Variations in isotope enrichments from different sources can be
substantial so the tables also include uncertainties. The cumulative
uncertainty for cytochrome-c (C560 H874 N148 O156 S4 Fe) is +-0.7 Da (560*.001
+ 874*.00007 + 148*.00007 + etc....). For larger proteins, the uncertainty
grows accordingly: HIV integrase 32330.0 +-1.8; Lamin A 74139.6 +- 4.0.
Questions:
Since these uncertainties are within the working precision and accuracy of many
modern mass spectrometers, what level of caution should we apply to our use of
average mass values? Has anyone measured the isotopic compositions of various
proteins, cells, whole organisms, etc...and if so, should we use these values
for our average mass calculations? I can imagine that the average mass of a
protein isolated from humans could be measurably different from the same protein
expressed in _E._coli_ and moreover depend on the bacterial growth conditions
(nutrient source, etc...).
Best regards to all...
Brett Phinney wrote:
> To all of the mass spec. experts,
>
> I have a question regarding the correct mass of several proteins and
> peptides that I have been using for calibration standards.
>
> One of the standards I have been using is Bovine Insulin oxidized beta
> chain. Sigma lists the mass as 3495.9 Bruker's calibration list also lists
> the monoisotopic mass as 3494.65. But when I pull the amino acid sequence
> from swiss-prot and Do the calculations on both Rockefellers web site and
> EMBL's web site, I get a monoisotopic mass of 3397.674. What could account
> for this mass difference? Am I doing something wrong?
>
> The other standard is Horse heart cytochrome C. Sigma lists the mass as
> 12384, Bruker lists the average mass as 12361.088 and I get an average
> calculated mass of 12374.02 including 1 Fe atom and 1 heme group with a mass
> of 616.6. Additionally, when I buy my standards from Sigma, for example,
> What modifications are on the cytochrome C. Swis-prot says cytochrome C has
> the following modifications
>
> Post translational modification: 1 ACETYLATION
> Binding site: 14 HEME (Covalent)
> Binding site: 17 HEME (Covalent)
> Binding site for metal ion: 18 IRON (Heme axial ligand)
> Binding site for metal ion: 80 IRON (Heme axial ligand)
>
> But the average mass with all of these modifications (calculated from
> Rockefeller's web site (www.proteimetrics.com)is 13088.50.
>
> Which mass is correct? and what am I actually getting from sigma?
>
> Thanks a lot in advance for the help
>
> __________________________________
> Brett Phinney
> North Carolina State University
> Department of Biochemistry
> Ph# 919-515-5786, Fax 919-515-2047
> ICQ# = 12442943
-- ________________________________ Steven H. Seeholzer, Ph.D. Staff Scientist Fox Chase Cancer Center 7701 Burholme Avenue Philadelphia, PA 19111phone: (215) 728-1111 fax: (215) 728-3574 email: sh_seeholzer@fccc.edu