Targeting Rare Proteins
A key challenge in biomarker discovery is identifying and quantifying typically low-abundance proteins in complex mixtures and at relatively high-throughput. Jeffrey S. Patrick, Ph.D., a scientist in the department of integrative biology at Eli Lilly (www.lilly.com), described the use of LC/MS/MS tools for multiple reaction monitoring to measure targeted protein biomarkers. The traditional global approach to biomarker discovery has evolved to a more targeted methodology in which researchers can zoom in on peaks of interest using an extracted ion chromatogram.
Comparing the advantages and limitations of ion trap MS versus triple quadrupole (QqQ) MS for targeted proteomics, Dr. Patrick pointed out that, while ion traps are sensitive and yield a broad range of protein fragmentation making them well-suited for protein identification, they have diminished selectivity. In contrast, QqQs offer “moderate sensitivity and outstanding selectivity,” with less noise and an enhanced, focused signal. They transmit only protein fragments of defined mass to the detector and are capable of multiplexed analysis of multiple proteins or peptides simultaneously, said Dr. Patrick.
As an example of a targeted proteomics strategy, Dr. Patrick described the quantification of orosomucoid (alpha-1-acid glycoprotein) in rat serum and its role as a biomarker for parathyroid hormone (PTH) activity and bone formation. PTH stimulates bone turnover, while GSK-3 inhibitor induces bone formation in osteopenic rats. Treatment with intermittent or continuous PTH or with GSK-3 inhibitor yielded a significant change in orosomucoid levels over time compared to control animals.
Dr. Patrick demonstrated how the use of internal peptide standards on ion trap MS allowed for rapid, rough quantification of orosomucoid peptides following trypsin digestion of bone extract. Orosomucoid was not detectable in bone extract using a global proteomics approach. In contrast, the targeted proteomics method enabled biomarker detection with a sensitivity down to about 50 ng/mL of protein.
Targeted methods also enable increased cycle times (15–30 minutes versus 120 minutes for global methods), quantification, and reduced sample needs (100 nL of serum for the orosomucoid experiments). They also eliminate the need for antibody development.
Focusing on proteomics sample preparation, Lisa Bradbury, R&D director for proteomics at Pall (www.pall.com), noted that because available “technologies are not robust,” attention to detail is crucial. Proteomics experimental design should include decisions regarding how to reduce the complexity of the sample (depletion, fractionation, digestion, tagging, affinity capture, and optional clean-up) and how to analyze the sample (LC-MS, LC-MS/MS, MS/MS, MALDI/SELDI MS, 2-D gel electrophoresis, FTMS).
She emphasized the importance of evaluating the reproducibility of methodologies upfront and deciding “where you can afford to lose proteins.” In general, with each additional step, reproducibility will decrease and loss of proteins will increase. However, how much will depend on the individual method used.
Decisions regarding proteomic methods should be made in the context of the biological questions and the sample set. “For some researchers, higher throughput is more important due to the number of samples that will be required for statistically significant data, while for others, identifying as many proteins as possible is important,” said Bradbury.
Bradbury discussed Pall’s Enchant™ kits, which deplete abundant proteins from serum or plasma samples. The second generation Enchant Multi-Protein Affinity Separations Kit is intended for use with human samples only and includes ligands for specific capture of IgG and albumin. One or both ligands can be used in a particular experiment in user-defined ratios.
The materials are disposable to minimize risk of cross-contamination. Starting sample sizes for dual albumin/IgG depletion are 5–50 µL of plasma or serum. Typical depletion efficiency is 97–99%. The kits can be used in the presence of denaturants added to release bound proteins. Percent depletion remains above 97% in the presence of urea or up to 3M of CHAPS. Alternatively, users can capture albumin on the column and then add denaturants, thereby isolating the albuminome in the eluant.