In the past decade, academic and industry researchers have successfully applied proteomic techniques, such as 2-D gel electrophoresis (2-DE), liquid chromatography (LC) and mass spectrometry (MS), to investigate protein-expression profile changes in different cell culture processes and conditions.
These proteomic techniques are useful tools to study mechanisms of action of conventional industrial cell culture methods to boost protein expression, such as hypothermal and hyperosmolality culture conditions. The well-established workflow of protein identification and relative quantification includes 2-DE analysis, spot selection and excision, tryptic digestion, followed by MALDI-TOF and tandem (MS/MS) mass spectrometry.
CHO, NS0 myeloma, and hybridomas have been studied for protein-expression profiles with hundreds of proteins identified to be up- or down-regulated in various functional groups, from structural proteins and endoplasmic reticulum chaperones to metabolic enzymes. These hits provide potential biomarkers and targets for rational cell line engineering approaches.
Compared to genomic methods, proteomic methods are lower throughput and more labor intensive. Some emerging new techniques in protein separation, identification, and quantification provide new tools for increasing throughput. 2D-Difference Gel Electrophoresis (2D-DIGE) labels up to three different protein extract samples with different fluorescent dyes, mixed and separated concurrently by 2-DE. The images from different dyes are merged, and the differences analyzed.
Quantitative MS methods that utilize stable isotope tags, such as ICAT (isotope-coded affinity tags), and isobaric amine specific tags, such as iTRAQ™, allow for relative protein quantity without the labor-intensive process of 2-DE. Up to four samples can be analyzed simultaneously by iTRAQ.
Robotic spot-picking has greatly increased the throughput of protein identification. In addition, regulatory proteins are often less abundant than structural proteins. New approaches such as membrane protein fractionation by surface biotinylation and enriched microsomal fractionation may capture changes in low-abundance protein expression and shed light on pathway studies.
As in other disciplines, analysis and interpretation of proteomic data in cell culture applications should always be coupled with genomic data and adequate biological replication. Follow-up and confirmatory studies using Western blotting and functional assays are essential to rule out false positives in the proteomic hits, leading to more successful identification of biomarkers for cell culture development.