Proteomics on the Bench Top: Rapid, Proteome-Wide Analysis

In proteomics, expression profiling allows a researcher to reveal the complex changes that underly normal biological responses and aberrant responses that underlie disease. The preparation, separation, measurement, and analysis of protein expression profiles has traditionally required prolonged and low-yield experiments by highly experienced staff.

Scientists working under Matthias Mann at the Max Plank Institute for Biochemistry have developed a method to easily screen the near-complete yeast proteome. Whole protein extracts are adsorbed to a pippette-tip filter and prepared in place. The use of pippette-based preparation steps minimizes sample waste and increases yield in addition to using methods that can be immediately applied in any lab.

Contaminants from cell lysis are removed with urea washes. Proteins were digested into fragment peptides using the LysC protease and alkylated in preparation for separation using reverse chromatography, where a polar mobile phase flows through a non-polar stationary phase. Prepared peptides were transferred from the treatment filter tip directly to a stage tip for loading onto the chromatograph. ¹

Peptide fragments are separated via reverse-phase ultra-high-pressure liquid chromatography (UHPLC). Data was obtained using a tandem liquid chromatography and mass spetromertry method (LC-MS/MS) using a Thermo Scientific Easy-nLC 1000 Liquid Chromatograph and sequenced with a Q Exactive Mass Spectrometer. A long, narrow chromatography column — 50 cm in length with a 75 µm inner diameter — is used to take advanatage of the increased resolution allowed by the very high pressure. The column was packed with fine 1.8 µm C-18 beads and eluted in an increasingly non-polar stationary phase.¹

In yeast and other model systems, Mann’s method offers a rapid and simple way to broadly detect proteomic changes. Previous methods have required the division of the sample into 80 or more fractions, greatly increasing required time and complexity.² A single run, requiring four hours using Mann’s method, can analyze the expression profile of more than 3,900 proteins in yeast, approaching the number thought to be expressed under laboratory conditions. Using this method, Nagaraj et al. were able to detect the decrease in translational pathways and increase in stress pathways brought about by heat shock.¹

Though a rapid, sensitive, and broad approach, this method may not be directly applicable to proteomics applications. A single-run covers a median of 23% of the proteome sequence and may be unable to distinguish changes in the proportion of protein isoforms or posttranslational modification of proteins.¹ Nevertheless, the advances in separation and detection are easily extended to more complex samples by adapting sample preparation, such as prefractionation, to enrich the sample in proteins of interest.

The simplicity and ease of Mann’s approach places bench-top proteomics into the hands of researchers working toward many biomedical goals. As pathways change in response to stress and disease, analysis of these pathways may give rise to clinically relevant biomarkers that will assist in the diagnosing and monitoring of many of today’s diseases. A similar method is being used to discover biomarkers indicating the presence and type of bladder and kidney cancers.³

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) (p. M111.013722)

2. Peng, J., et al. (2006) ‘Evaluation of multidimensional chromatography coupled with tandem mass spectrometry (LC/LC-MS/MS) for large-scale protein analysis: the yeast proteome‘, Journal of Proteome Research, 2 (1), (pp. 43-50)

3. Lin, L., et al. (2012) ‘LC-MS based serum metabolic profiling for genitourinary cancer classification and cancer type-specific biomarker discovery‘, Proteomics, 12 (14), (pp. 2238-2246)