Intact Mass Determination

Intact mass determination includes several sample prep procedures (outlined in the figure below), including buffer exchange, solid-phase extraction, de-glycosylation, and limited proteolysis. Prior to introduction to the mass spectrometer, proteins are separated by liquid chromatography under either denaturing or native conditions along with UV detection. We then analyze intact mass of proteins using the Eclipse Orbitrap mass spectrometer. Finally, we process the data using BioPharma Finder software.

Diagram showing intact protein mass analysis workflow, including sample preparation, LC-UV separation, MS/MS detection using Orbitrap, and final data analysis.

Figure 1. Workflow for Intact mass LC-MS analysis

Multi-panel figure showing denaturing SEC-UV, Orbitrap Eclipse PTCR-MS, sliding window deconvolution, raw mass spectrum with charge states, and final deconvoluted mass results.

Figure 2. High resolution determination of intact mass in denaturing and native conditions. (A) Rapid desalting of proteins coupled to Orbitrap MS analysis allows robust intact mass profiling of a variety of protein samples. (B) Raw spectra of intact proteins generated by electrospray ionization (ESI-MS) generated ionic signals (m/z), corresponding to a series of successive charge states in which the detected proteoforms are redundantly represented. (C) Deconvolution results show a discrete series of intact mass peaks which correspond to a distribution of 0-2 glycations present on denatured ConA monomer.