Evaluation of Protein Level Separation as Sample Preparation for Absolute Protein Quantification by LC-MS/MS, presented at the 59th ASMS conference on Mass Spectrometry and Allied Topics, June 5-9, 2011 in Denver, CO.

Introduction: Absolute quantification of proteins has traditionally been done using ligand binding assays. While such methods have been developed and used routinely with great success, there are limitations. Two of the most important limitations are selectivity, and the time required to develop selective and rugged antibody reagents and assays. LC-MS/MS approaches that do not require antibodies are increasingly finding utility as an early supplement to ligand binding assays. The two biggest issues with LC-MS/MS quantification of proteins are obtaining very low detection limits and the required selectivity in SRM mode in the presence of background protein. The work presented here aims to understand how separation at the intact protein level might affect the LLOQ of quantitative assays based on LC-MS/MS.

Methods: Quantitative assays based on surrogate tryptic peptides for several human UGT isoforms in whole microsomal protein fractions have been developed. Separation at the protein level is done using the Gelfree 8100 one-dimenstional gel electrophoresis system (Protein Discovery). The system is set up so that the molecular weight fraction from 55 to 70 kda is collected in liquid phase for digestion. SRM detection of unique tryptic peptides for each UGT isoform was performed using an AB Sciex 5500Qtrapmass spectrometer coupled to an Eksigent Express HT Ultra HPLC. Peptide separation was effected with a 1 mm Jupiter Proteo Column (Phenomenex). Stable isotopically-labeled internal peptide standards are used with recombinant protein standards to build calibration curves for quantification of UGTs in liver microsomes.

Preliminary Data: The original methods for UGT quantification use peptide detection in SRM mode. The methods showed useful detection limits ranging from 0.5 to 5 pmol/mg of microsomes depending on the specific isoform. However, it is desirable to extend these quantification limits at least another order of magnitude (0.05 pmol/mg). The LOQ of the current method is limited in these assays by the background in SRM. SRM, while highly selective for small molecules, for proteins, does not have the selectivity required to provide the near zero background typical of small molecule SRM. In order to reduce the background, gel electrophoresis is used to fractionate the microsomal protein sample prior to digestion with trypsin. Preliminary results show background levels in SRM are reduced by 10 to 20 fold depending on the peptide monitored. Additionally, use of the Gelfree 8100 allows the use of 100 ul of microsomal protein at 20 mg/ml concentration compared to the 10 ul used for the current methods. Taken together, the current indications are that this approach will allow us to reach levels below the current best of 0.5 pmol/ml. Accuracy and precision need to be established using this approach before actual LLOQ values can be determined and reported.

Novel Aspect: Fraction collection from tube gels as sample preparation for quantification of human UGT isoforms by LC-MS/MS.


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