Using Targeted Proteomics with an UltraFast Triple Quadrupole Mass Spectrometer to Confirm Protein Overexpression

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Protein overexpression is an experimental approach that is utilized in a variety of fields of research. Protein expression is most commonly confirmed with Western blotting, but this technique requires an antibody to detect each target protein. However, protein expression can be confirmed by targeted proteomics using a triple quadrupole mass spectrometer in MRM mode to selectively detect and quantify peptides produced by trypsin digestion of the target protein. In this article, targeted proteomics was used to confirm protein expression by using an E. coli genetically modified to overexpress phosphoglucokinase (Pgk). The trypsin-digested samples used in the analysis were prepared by a proven experimental protocol. The targeted proteomics software Skyline was used to automatically create 110 MRM transitions that were based on the amino-acid sequence of Pgk, which it then exported to an LCMS-8060. With this data, the nano-LC- triple quadrupole mass spectrometer (LCMS-8060) was able to perform measurements that confirmed Pgk overexpression.

Introduction

Proteins undertake a wide range of functions in living organisms. Protein overexpression is an experimental strategy with many uses, including elucidating protein function in basic research, constructing modified microorganisms for bioproduction, and the biopharmaceutical production of antibodies and other proteins. Confirming whether overexpression has been achieved requires the measurement of protein expression levels, which are typically measured by Western blotting. However, this technique requires an antibody to detect each targeted protein. While antibodies for a variety of proteins are commercially available for use in basic medical research, in the field of metabolic engineering, genes from non- model animals are overexpressed in microorganisms and require the preparation of an antibody for each target protein.

December 11, 2023 GMT