Reliable Proteomic Analysis
The holy grail of protein profiling is to determine comprehensively the identity, abundance, and isoforms of proteins in a large number of clinical samples, maintains Jun Qu, Ph.D, associate professor at the University of Buffalo, SUNY.
“Biomedical discovery necessitates comparison of multiple samples to overcome known problems with individual variability. However, accurate and reliable profiling of multiple samples remains challenging,” he explains.
“We developed a highly reproducible and quantitative method that can detect proteomic signatures despite a highly variable biological background.”
The “precipitation/on-pellet-digestion” method relies on a unique detergent cocktail to achieve a near complete extraction, including that of membrane proteins. Addition of organic solvents, like acetone, denatures and aggregates the pellet, which is notoriously difficult to re-solubilize.
“Our sample prep philosophy is the simpler, the better,” continues Dr. Qu. “We let the enzymes digest the proteins right on the pellet. This method produces a clean peptide mixture suitable for LC-MS analysis.”
The team designed a unique separation column that features low void volume, large loading capacity, and high separation efficiencies. Peptides are separated on a heated, 100-cm-long column during a seven-hour run. The team determined that a label-free MS approach was the most cost-efficient method. It provided excellent accuracy for relative protein quantitation when well controlled.
The process was previously applied to several clinically important questions, such as finding underlying causes of hibernating myocardium and chronic obstructive pulmonary disease. The most recent research focused on HIV.
“Five percent of HIV carriers never progress to a disease state,” says Xiaomeng Shen, a Ph.D. candidate in Dr Qu’s lab. “Proteomic comparison of normal progressors vs. long-term nonprogressors (LTNPs) provides highly valuable insights for directing therapeutic efforts.”
Comparison of 10 individuals from each cohort resulted in the identification of 78 differentially expressed proteins. Subsequent annotation by Ingenuity Pathway Analysis software pointed to several signaling pathways that may account for viral control mechanisms in LTNPs.
“We were excited to find the SAMHD1 protein in the interferon pathway, which was recently implicated as a critical player in viral restriction,” remarks Dr. Qu, who notes that it would take many years to generate the same amount of data by the traditional methods.