In label free quantitation (1), protein profiling comparisons are based on the relative intensities of extracted ion chromatograms from complex samples such as enzymatic digests. This approach therefore, does not require any metabolic, chemical, enzymatic labeling or premixing of the samples to be compared Multiple datasets, typically run in triplicate, are aligned using mass and LC elution time. The approach requires excellent mass accuracy and reproducible chromatography, thus, the mass spectrometer platforms of choice for this procedure are the LTQ Orbitrap XL and the Bruker Apex Qe 9.4T FT-ICR. Each of these mass spectrometric platforms routinely perform with mass accuracies of better than 3 ppm. The LTQ Orbitrap XL is equipped with a Waters nanoACQUITY which has superb chromatography reproducibility of ±0.2% minute elution time (2). Data analysis is done with the Keck Biostatistics Resource under the direction of Dr. Hongyu Zhao.
- Recommended sample amount is 5 to 25µg; each run is done on 0.2µg
- Old., W.M., Meyer-Arendt, K., Aveline-wolf, L., Pierce, K.G., Mendoza, A., Sevinsky, J.R., Resing, K.A., and Ahn, N.G. (2005) Comparison of Label-free methods for Quantifying Human Proteins by Shotgun Proteomics. Mol Cell Proteomics, 4(10):1487-1502.
- Stone, K.L. and Williams, K.R (2009b) Reverse-Phase HPLC Separation of Enzymatic Digests of Proteins in The Protein Protocols Handbook, 3rd edition (Walker, J., ed.) 937-946.