Skip to Main Content
Proteomics
YSM Home

INFORMATION FOR

Electrospray Mass Spectrometry

  • Routine molecular weight determination of synthesis products, oligos & other biomolecules
  • Mass determination of synthetic chemical intermediates (exact mass is done on the FT-ICR)
  • Protein mass determination ∼ 100ppm mass accuracy (100 to 100,000Da)
  • Information on sample purity
  • Location of protein post translational modifications

Important Links

  • Incorporation of substituted amino acids (i.e. selenomethionine)
  • Analysis of oligonucleotides up to 90 mers
  • Manual MS/MS sequence analysis
  • Location of sites of cross linking
  • LC-MS/MS analysis of simple and complex protein mixtures

Sample Preparation: Protein/peptide solutions in 10 μM volatile buffers can sometimes be analyzed directly while samples with higher salt concentrations require prior C18 or C4 ZipTip desalting


ESMS
Bottom spectra is the mass of the native protein (28,871Da);
Top spectra is of the substituted selenomethionine protein (29,296Da)
for incorporation of 9 selenomethionines (Waters Q-Tof Micro)

Impact of Multiply Charged Ions on Mass Spectrometry

Although we sometimes see +2 charged species during MALDI-MS, short peptides tend primarily to give just the +1 species so this makes it easy to directly interpret MALDI-MS spectra. The problem is that all types of MS actually measure the mass/charge ratio (m/z) as opposed to the mass. Hence the following two scenarios give identical spectra with a single observed peak (in positive ion mode) at m/z of 2,001:

Actual (M) Peptide mass = 2,000, Charge = +1, Observed (M+H) m/z = (2,000 + 1)/1 = 2,001

Actual (M) Peptide mass = 4,000, Charge = +2, Observed (M+H) m/z = (4,000 + 2)/2 = 2,001

Multiple charge states are potentially a severe problem for electrospray ionization mass spectrometers. Instead of the single (M+H) species characteristic of linear MALDI-MS, electrospray usually gives a broad spectrum of multiply charged ions for each species present, which is why a mass spectrometer equipped with an electrospray source that has an upper m/z limit of 1,800 can easily determine the m/z for a 50,000 dalton protein. Because of multiple charging many m/z ratios obtained from the electrospray ionization masses.