Advances in Single-Shot Protein Expression Profiling

An organism expresses thousands of proteins simultaneously. The profile of this expression gives a researcher insight into the inner workings of an organism during health and disease. Many existing methods to monitor the entire proteome require experienced staff, extensive time investment and complex, drawn-out methodology, preventing many labs from routinely performing proteome-wide screens. Recent method developments produce robust results from single experiments while reducing time and expertise required.

Proteome-Wide Coverage in Four Hours

Researchers in Matthias Mann’s lab at the Max Plank Institute for Biochemistry have developed a simple, rapid single-shot method for proteome wide screen verified in a yeast model system. The method expands earlier work by using simplified sample preparation and instrumentation, making the method suitable for most research and clinical labs. Protein isolated from the sample is prepared using only pipette-based treatments. Protein expression levels are identified by separating peptides using ultra high-pressure liquid chromatography (UHPLC) on a Fisher Thermo Easy nLC-1000. The eluted peptides are selected and sequenced using a Fisher Thermo Q Exactive mass spectrometer.

A single-shot experiment requires only four hours of processing and reliably detects upwards of 92% of expressed proteins per run. This method requires only four hours and minimal maintenance or set-up between runs. Samples containing only a few micrograms of protein are able to produce robust expression profiles.

Applications to Research and Translational Medicine

Mann’s method was able to detect the alteration in translational and stress pathways that accompany heat shock in yeast. While the method has a broad enough coverage to detect changes in the expression of pathways or single proteins, it lacks the resolution to detect fine-scale changes such as post-translational modifications and isoform ratios.

Though Mann’s method does not require fractionating the sample into multiple parts, it is equally applicable to more complex samples from mammalian and human tissue relevant to clinical research. The four-hour process time and minimal changes required in the setup allow rapid processing of complex samples and repeition for statistical power. Variations in sample handling and processing account for large variances that are a hindrance to proteomic research. Simple methods produce powerful results.

References

1. Nagaraj, N., et al. (2011) ‘System-wide Perturbation Analysis with Nearly Complete Coverage of the Yeast Proteome by Single-shot Ultra HPLC Runs on a Bench Top Orbitrap’, Molecular and Cellular Proteomics. 11 (3).