Posters Highlight Advanced Methods
Many of the posters presented at the ASMS meeting described studies utilizing innovative MS methods such as multiple reaction monitoring (MRM)-MS, differential MS (dMS), and label-free quantitative LC/MS-based analysis. These techniques are expanding the capabilities and market opportunities for MS technology, but they also create new challenges for sample preparation, fractionation, and high-abundance protein depletion upstream of MS.
In the poster “MRM for Oral Cancer Biomarker Validation in Saliva: Inherent Challenges, Solutions and Methods Development,” Ebbing de Jong and colleagues from the University of Minnesota describe efforts to simplify sample prep for MRM validation studies of protein biomarkers in saliva through amylase depletion.
The researchers loaded saliva samples onto starch columns to remove much of the amylase component, and then eluted the remaining protein fractions. These were run on Western blots or digested and analyzed by 1-D LC-MS on a Thermo Scientific LTQ Orbitrap. A comparison of the amylase-depleted sample versus whole saliva showed that amylase depletion revealed new protein bands and allowed for enhanced detection of low-abundance peptides such as kallikrein-1 on MRM chromatograms.
A.K. Yocum and colleagues from the University of Michigan described the development and optimization of an assay to discriminate between benign and cancerous prostate cell lines using multiplexed stable isotope dilution MRM-MS/MS.
They presented the results of a study to measure absolute concentrations of eight peptide biomarkers for prostate cancer in whole cell protein extracts. The protein extracts from six benign and cancerous prostate cell lines were separated by 1-D SDS-PAGE and digested, and the peptides were analyzed using LC-MRM-MS/MS on the 1200 Series HPLC-CHiP and 6410 Triple Quadrupole from Agilent Technologies.
Based on the results, the authors concluded that the multiplexed MRM-MS/MS assay will provide greater discriminatory power for the identification of tissue samples positive for prostate cancer, reducing the frequency of false positive findings and unnecessary biopsies.
Researchers from Pacific Northwest Laboratory and Oregon Health Sciences University presented the poster, “Application of Label-Free Quantitative LC-MS-based Proteomics for Biomarker Identification in Salmonella typhimurium,” in which they identified a set of proteins coordinately upregulated on infection of host macrophages.
The study involved a comparison of the proteomes of knock-out mutants of Salmonella, in which proteins associated with regulating virulence are not expressed, with the proteomes of wild-type Salmonella, to identify specific proteins involved in infection that could serve as novel targets for drug discovery.
Samples were fractionated and analyzed by LC-MS/MS on a Thermo Scientific LTQ Orbitrap, and candidate protein biomarkers were selected based on the following inclusion criteria: must exhibit > twofold reduction in expression in mutant compared to wild-type; must be coordinately regulated in a minimum of seven of twelve regulator mutants; and must be identified by > three peptides. Five LC-MS discovered biomarkers were validated via Western blot, which demonstrated upregulated expression on Salmonella infection of host macrophage cells.