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Small Protein MS

Small proteins, mass of 25 kDa or less, are small enough to be studied by normal resolution (nominal mass) and high resolution MS analyses.

Nominal mass MS

The typical accuracy of nominal mass MS data is to the first decimal place (0.1 m/z). Typical instruments include quadrupole and ion trap mass spectrometers. The standard ProMass deconvolution algorithm (ZNova) deconvolutes nominal mass MS data to confirm protein average isotopic mass, determine amino acid loss or addition, and identify modifications with mass differences greater than 1 Da. The data below are from horse myoglobin with an expected average isotopic mass of 16951.4 Da. The ZNova algorithm can also be applied to high-resolution MS data from Orbitraps or Q-ToFs if average mass data is preferred over exact mass.

Raw ESI spectrum of horse myoglobin generated on a Waters Xevo G2-XS Q-ToF coupled to an Acquity I-Class UPLC.

ProMass removes the background signal observed in the raw ESI spectrum to generate a cleaner spectrum for deconvolution. The charge states (m/z) for each ion are labeled in blue, with masses shown to the first decimal place.

ProMass deconvolutes the multiple charge states to generate the observed neutral mass (16951.2 Da). Since the expected exact mass is 16951.4 Da, the observed mass error is -0.2 Da (-0.0012% or -12 ppm).

High Resolution MS (HRMS)

Deconvolution of the isotopically-resolved HRMS data allows amino acid composition to be confirmed by using the exact mass of the deconvoluted ESI spectrum to confirm the chemical formula of the protein. For example, mass accuracy of ±0.0005% (± 0.05 Da in 10 kDa) can identify a single glutamine to glutamic acid substitution difference in the intact protein mass. The accuracy of the deisotoped mass spectrum is consistent with the calibrated accuracy of the instrument, which is typically 5 ppm or less for HRAM instruments. The data below are from horse myoglobin which has an exact mass of 16940.965 Da.

Raw ESI spectrum of horse myoglobin generated on a Waters Xevo G2-XS Q-ToF coupled to an Acquity I-Class UPLC.

The background subtracted ESI spectrum removes the small bump of background signal observed below the protein ion signal. The charge states (m/z) for each ion are labeled in blue, with masses shown to the fourth decimal place.

ProMass deisotopes the observed ions in the background subtracted spectrum. After deisotoping, ProMass deconvolutes the multiple charge states to generate the observed neutral charge exact mass (16941.031 Da). Since the expected exact mass is 16940.965 Da, the observed mass error is 0.066 Da (0.00039% or 3.9 ppm).