Biomolecule mass determination by Mass Spectrometry

Principles

Masses of molecules of interest (proteins with MW<100 kDa or peptides) can be determined with high accuracy using mass spectrometry. The size of a known protein therefore or peptide can be confirmed. Isoforms of the molecules may also be detected, however in-depth analysis (i.e. character and site of Post Translational Modifications) is not provided.

Sample

The sample could be in solid form (recommended) or in soluble form, free of detergents or salts using an appropriate solvent (ammonium acetate ). Information on sample purity, expected MW, buffer and other components in solution and sample stability (T oC, possible denaturation / aggregation in high concentration of acetonitrile) are wanted.

Instrumentation

ESI generates multiple charge states of proteins. LTQ Orbitrap XL ETD is accurate within 1-5 ppm for proteins and mass range m/z 200-4000.

Experiment

Sample introduction by ESI is performed by direct injection using a syringe pump.

Data Analysis

Mass is calculated by mathematical transformation of the ion series using Promass Deconvolution 2.5 (Thermo Scientific). Manual validation is also required.

Results

A written report is provided describing the analysis undertaken and masses measured (spectrum included ). Where appropriate, comparisons with calculated masses are made. The report is sent sent by e-mail. Raw data are archived by ProFI. If the data are used as a part of a scientific publication, reference to ProFI Lab should be made in the acknowledgements.

Time required for intact protein analysis is sample dependent. Please have a look at the examples.

Some proteins may not be able to be analyzed (poor ionization, instability etc)