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Small Protein MS

Small proteins, mass of 25 kDa or less, are small enough to be studied by 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). ProMass deconvolutes nominal mass MS data to confirm protein 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 nominal mass of 16951.4 Da.

Raw ESI spectrum of horse myoglobin generated on a Waters Xevo X2 qTOF 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 charge exact 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 HRMS data allows amino acid composition to be confirmed by using the exact mass of the deconvoluted ESI spectrum to determine 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 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 X2 qTOF 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).