****************************************************************************** From: Brett Phinney Date: Fri, 14 Feb 1997 13:40:45 -0800 Subject: peptide mass calculations Organization: University of Texas at Austin I am doing a peptide map of a protein I am interested in. For some preliminary data I am first doing a trypsin digest and then I am using a mass spec. to get the mass of my fragments, with the hope that I will be able to determine disulfide bond locations. My question is that when I calculate the expected mass of my fragments I can get differing results if I calculate the mass using the average mass or the monoisotropic mass. Does anyone have any suggestions as to which calculation method I should use? Does anyone have a good reference on this subject? Thanks a lot Brett Phinney ****************************************************************************** From: vanreet@ilink.nis.za (Teunis vanRee) Date: 15 Feb 1997 08:19:19 GMT Subject: Re: peptide mass calculations Organization: Network Information Services There's no way you can use the average mass no. The mass spectrometer sees only monoisotopic ions. So calculate the monoisotopic mass nos. Regards ***************************************************************************** From: mrdio007@aol.com (MrDio007) Date: 16 Feb 1997 20:57:26 GMT Subject: Re: peptide mass calculations Organization: AOL http://www.aol.com I think Fenseleau wrote a good one in Analytical Chemistry within the last ten years. Dick Smith's group might have done one as well. This question is best answered there but,... I think it depends upon the mass of the pieces you are looking for. matt sweeney ****************************************************************************** ****************************************************************************** From: Stuart Thomson Date: Mon, 17 Feb 1997 13:02:18 +1100 Subject: Re: peptide mass calculations Organization: Monash University } There's no way you can use the average mass no. The mass spectrometer sees } only monoisotopic ions. So calculate the monoisotopic mass nos. } } Regards I Beg to differ. This question really is, does the resolution of my mass analyser allow the differentiation of the full isotopic distribution at the mass in question? If not then use average masses, as the mass from the mass spec will be such. If so the use monoisotopic masses as the mass from the mass spec will be so. So it's horses for courses Brett, if you can see the isotopic distribution of your molecular ion for your tryptic peptides, then use those masses, if not don't as you'll be out by a couple of mass units. Cheers Stuart Thomson. ****************************************************************************** From: Kurt Benkestock Date: Mon, 17 Feb 1997 10:46:38 +0100 Subject: Re: peptide mass calculations Organization: Pharmacia & Upjohn It depends on which resolution you have on the mass spectrometer during the analysis. On a quadrupole instrument where the resolution could be maximized to 1000 you have to use both the mono- and the average molecular mass for the calculation. For fragments upp to around 1200-1400 Da monoisotopic mass can be used. Above this value the resolution is not sufficient to resolve the monoisotopic peaks. As a result, you get one broad peak and the average mol mass must be used. Good Luck Kurt Benkestock ****************************************************************************** ****************************************************************************** From: PAREESDM@apci.com Date: Mon, 17 Feb 1997 17:26:29 -0500 Subject: Re: peptide mass calculations Organization: * If I understand the question correctly, the answer is dependent on the resolution of the mass spectrometer that you are using. If the instrument has sufficient resolution to resolve the isotopic clusters of the molecular ions, then the masses you will obtain are the isotopic masses and those are the calculated ones to use for comparison. If your instrument has insufficient resolution (e. g., an old-style, non-delayed extraction, non-reflecting time-of-flight used for matrix-assisted laser desorption/ionization of species with masses in excess of a few hundred daltons), then the molecular ion clusters may be non-resolved and the masses measured would be the average masses; a simple combination of all of the isotopes of all of the elements present, taking into account their relative abundances. In this case, the data should be compared to isotopic average molecular weights (plus any cationization that your technique may incorporate). Dave Parees My employer accepts no responsibility for my internet activity ****************************************************************************** ****************************************************************************** From: James Pavlovich Date: Tue, 18 Feb 1997 09:27:28 -0800 Subject: Re: peptide mass calculations Organization: University of California, Santa Barbara It depends on a couple of factors which is the best value to use. In your case, tryptic fragments are relatively small so using the monoisotopic mass would be most apprpriate. Generally speaking you should use the monoisotopic mass so long as your instrument has sufficient resolution to distinguish the individual isotopic masses (alway 1 amu apart). However, at mass above ~1500 amu the monoisotopic mass become decreasingly abundant and will eventually be insignificant at masses on the order of several thousand amu. At that point, average mass is more meaningfull even if you have the resolution. Also note that for multiply-charged species produced by electrospray ionization, the isotopic peaks will be closer together by 1/z, and require correspondingly higher resolution to distinguish the isotope peaks. Therefore, use of average mass may be more meaningful when calculating mass from highly charged species, unless you have a very high res instrument. Good luck, James Pavlovich -- ____________________________________________________________________ ____________________________________________________________________ James G. Pavlovich, Ph.D. Mass Spectrometry Facility Phone: 805-893-4252 Chemistry Department FAX: 805-893-4120 University of California Email: pavlovic@chem.ucsb.edu Santa Barbara, CA 93106 ____________________________________________________________________ ____________________________________________________________________ ****************************************************************************** From: sumner@chemvx.chem.tamu.edu (Sumner Lloyd) Date: Tue, 18 Feb 1997 15:03:20 -0600 Subject: Average/Monoisotopic Organization: * Brett, The mass that you should use is dependent upon the resolving power of the mass spectrometer that you are using. If you have sufficient resolution to separate the isotopes of the ion peak in question than you should use the monoisotopic masses. On the other hand, if the resolving power of the MS is low such as MALDI linear time-of-flight, than you should use the average mass because what you observe in the mass spectrum is the unresolved isotopic profile that is centroided at the average mass. Another suggestion for your interest in disulfide bonds is the alkylation of disulfides with methyl or ethyl iodide for the primary determination of the number of disulfide bonds. The number of disulfides is determined by shifts in the mass of the suspect protein as determined by MALDI or ESI. Further, higher order structures can be investigated by selective alkylation of disulfide bonds and their effect on structure. Best Regards, Lloyd W. Sumner, Ph.D. Associate Director, The Laboratory for Biological Mass Spectrometry Department of Chemistry Texas A&M University College Station, TX 77843-3255 Voice #(409)845-8404 Facsimile#(409)845-4719 Email Sumner@chemvx.tamu.edu ******************************************************************************