Mass Spectrometry - AIMS Lab

ElectroSpray Ionization Mass Spectrometry (ESIMS)

  • Samples must be free from phosphate buffers and high salt contamination (Sodium and Potassium salts, e.g.) in order to avoid capillary and skimmer contamination and blockage. Also, the presence of tfa will inhibit the formation of ions from peptides. If an acid is necessary, use about 0.1% formic or acetic acid. DO NOT USE TFA. Avoid GLYCEROL as it will also inhibit the ionization of peptides and proteins.
  • Large amounts of any easily ionized impurity (alkyl ammonium salts, triethylamine for example) are invisible to UV detectors, but will reduce or suppress the ionization/observation of the analyte of interest in the Electrospray Ionization process. All buffers are best removed before Electrospray Ionization. If buffers must be present, use organic buffers at a concentration of no more than 10 . 20mM. This applies to NaCl or KCl as well.
  • I suggest that protein or peptide samples be supplied in aqueous solution (HPLC grade) at a concentration of ca. 30 to 80 pmol/uL (30 . 80uM). The total volume should be about 20uL. The solutions must be free of particulate matter.filter if necessary and any buffers / salts present should be noted on the sample submission form.
  • If low MW compounds are your game, then a small amount (100-300 ug) of neat compound in a small container must be supplied, or a ~10 uM solution.
  • Peptides of sufficient size and proteins will exhibit a series of peaks corresponding to multiply protonated ions formed in the electrospray process (~ 1 proton per kDa) and these peaks must be transformed to give the molecular weight. Protein solutions may be run through a Sephadex column to separate the protein from any buffers prior to ESI. Generally, a protein sample will be run in 1:1 methanol/water with 0.1% formic acid and about 10 to 25 pmol of protein will be used per measurement.
  • In general, the Electrospray process evaporates ions from solution where they are transmitted via a series of lenses into a mass analyzer, producing a mass spectrum. The ionization process is very gentle and often produces but a single ionic species characteristic of the molecule of interest. For neutral low molecular weight species, the ion is the protonated molecule, MH+ ; however, for this to be formed, the molecule must be capable of protonation. If sodium or potassium salts are present, then a sodiated or potassiated molecule may be formed.
  • Low molecular weight salts . quaternary amine compounds for example . will afford the cation in the positive mode and the anion in the negative mode of operation.