Proteomics

What for? Disease targets Gene finding Secondary modifications Measuring expression levels Protein-protein interactions

Whats new? Mass spectrometry was invented turn of century (Thomson) Noble price to Aston 1930s MALDI-TOF (Henzel et al, 1993) Nano-electro-spray (Wilm, Mann 1996s) coupled to tandem mass spectrometer

M + e- => M+· + 2e-

MALDImatrix assisted laser desorption ionization TOFtime-of-flight

Positive ESI-MS m/z spectrum of the protein hen egg white lysozyme. The sample was analysed in a solution of 1:1 (v/v) acetonitrile : 0.1% aqueous formic acid and the m/z spectrum shows a Gaussian-type distribution of multiply charged ions ranging from m/z to Each peak represents the intact protein molecule carrying a different number of charges (protons). The peak width is greater than that of the singly charged ions seen in the leucine enkephalin spectrum, as the isotopes associated with these multiply charged ions are not clearly resolved as they were in the case of the singly charged ions. The individual peaks in the multiply charged series become closer together at lower m/z values and, because the molecular weight is the same for all of the peaks, those with more charges appear at lower m/z values than do those with fewer charges (M. Mann, C. K. Meng, J. B. Fenn, Anal. Chem., 1989, 61, 1702). The m/z values can be expressed as follows:m/z = (MW + nH+) n where m/z = the mass-to-charge ratio marked on the abscissa of the spectrum; MW = the molecular weight of the sample n = the integer number of charges on the ions H = the mass of a proton = Da. If the number of charges on an ion is known, then it is simply a matter of reading the m/z value from the spectrum and solving the above equation to determine the molecular weight of the sample. Usually the number of charges is not known, but can be calculated if the assumption is made that any two adjacent members in the series of multiply charged ions differ by one charge. For example, if the ions appearing at m/z in the lysozyme spectrum have “n” charges, then the ions at m/z will have “n+1” charges, and the above equation can be written again for these two ions: = (MW + nH+)and = (MW + (n+1)H+) n(n+1) These simultaneous equations can be rearranged to exclude the MW term: n(1431.6) –nH+=(n+1) – (n+1)H+ and so:n(1431.6)=n(1301.4) – H+ therefore: n( )= – H+ and:n=( H+) ( – ) hence the number of charges on the ions at m/z = = Putting the value of n back into the equation: = (MW + nH+) n gives x 10=MW + (10 x 1.008) and soMW=14,316 – thereforeMW=14,305.9 Da The observed molecular weight is in good agreement with the theoretical molecular weight of hen egg lysozyme (based on average atomic masses) of Da. This may seem long-winded but fortunately the molecular weight of the sample can be calculated automatically, or at least semi- automatically, by the processing software associated with the mass spectrometer. This is of great help for multi-component mixture analysis where the m/z spectrum may well contain several overlapping series of multiply charged ions, with each component exhibiting completely different charge states. Using electrospray or nanospray ionisation, a mass accuracy of within 0.01% of the molecular weight should be achievable, which in this case represents +/- 1.4 Da.

Software Produced at NIH: Protein digestion softwareProtein digestion software (SMSF, NIDDK) The NCBI protein database (NCBI, NLM) Identify a protein based on its molecular weight (SMSF, NIDDK) Molecular mass/formula and electrospray calculators (SMSF, NIDDK) Flicker comparison of 2D gel images across the Internet (NCI, Frederick) 2DWG meta-database of 2D protein gel images found on the Internet (NCI, Frederick) Table of links to 2D gel electrophoresis database servers on the Web (NCI, Frederick) 2D protein gel image and spreadsheet databases using the dbEngine web search engine (NCI, Frederick) The NCBI protein database Identify a protein based on its molecular weight Molecular mass/formula and electrospray calculators Flicker comparison of 2D gel images across the Internet 2DWG meta-database of 2D protein gel images found on the Internet Table of links to 2D gel electrophoresis database servers on the Web 2D protein gel image and spreadsheet databases using the dbEngine web search engine Software Available Elsewhere: PROWLPROWL....protein identification tools from Rockefeller University Mascot....protein digest and identification software ProteinProspector....proteomics tools for mining sequence databases in conjunction with MS Canadian mirror of the expasy tools....many pieces of software for proteomics research...a faster site AAcompIdent....identify a protein from its amino acid composition AACompSim....identify proteins with similar amino acid composition Compute pI/Mw....theoretical PI and molecular weight cacluations for a protein FindMod....predict post-translational modifications and find single aa substitutions in peptides MOWSE....identify proteins from peptide digest masses MultiIdent....identify proteins from pI, molecular weight and aa compositions PeptIdent....identify proteins from pI, molecular weight and peptide mass fingerprinting PeptideMass....computes masses of peptides from an enzyme digest on a protein ProFound....protein identification by peptide mapping TagIdent....identify a protein by pI and molecular weight, or by sequence tag (e.g. 6 aa's in sequence) Prediction servers at CBS....prediction of glycosylation sites on proteins, etc A Mass Spectrometry and Biotechnology Software Resource....links to many software packages Packages of software from an Australian laboratory Deltamass....A listing of mass differences caused by modifications to aa or aa substitutions Deltamass....A listing of mass differences caused by modifications to aa or aa substitutions iProClass....software for retrieving/matching protein sequences Mascot ProteinProspector Canadian mirror of the expasy tools AAcompIdent AACompSim Compute pI/Mw FindMod MOWSE MultiIdent PeptIdent PeptideMass ProFound TagIdent Prediction servers at CBS A Mass Spectrometry and Biotechnology Software Resource Packages of software from an Australian laboratory Deltamass iProClass