Preparative Electrophoresis of Clinical Samples for Analysis by Mass Spectrometry
Wed 8:00 AM - Track 1: Sample Preparation Strategies
Jeremy Norris
Protein Discovery
Jeremy Norris, Protein Discovery, Inc., 418 S Gay St, Knoxville, TN 37902-1142
Recently, we have developed strategies that utilize the technique of polyacrylamide gel electrophoresis as a means to accomplish sample preparations of clinical samples for analysis by mass spectrometry. This approach has the advantages of being easily automated, preparative, and specific. Furthermore, the technique is unique to many other preparative techniques because the separation provides molecular weight information about the sample.

Gel-based separations are carried out in a tubular format with the samples eluting from the tube gel in order of their corresponding electrophoretic mobility. The liquid fraction resulting from these separations are collected for analysis using mass spectrometry. Specifically, these separations are provided in two formats: 8-channel and 96-channel format for medium-to-high throughput applications.

Applications of this approach for the processing of clinical samples for quantitative analysis by LC/MS/MS will be discussed. In one example, native gel electrophoresis is used to selectively enrich endogenous peptides from plasma samples. The advantages include removal of highly abundant proteins from the samples and neutral species such as phospholipids from the sample. For comparison, samples prepared using traditional techniques also analyzed. Analysis of electrophoretically processed samples indicate phospholipid exclusion >99.5%. The analysis of angiotensin I from human plasma was performed using the techniques described above. Sensitivity of the assay was less than 1 ng/mL, allowing for the quantitation of endogenous levels of angiotensin I.