|Faculty:Ye (Tony) Hu, Ph.D., Jason Sakamoto, Ph.D., Haifa Shen, Ph.D., Ennio Tasciotti, Ph.D., Arturas Ziemys, Ph.D.|
Problem: The latest cancer statistics published by the American Cancer Society suggest a downward trend in cancer death rates for many of the most prolific killers, such as lung, colorectal, prostate, and breast cancers. Significant contributors to this marked decline in mortality rate can be attributed to patient education, improved access to healthcare, and technological advancements in early detection modalities/procedures. With regard to the latter, the scientific community has been exploring innovative strategies to detect subtle biological changes that are associated with the initiation of, or precursors to, pathologic development. In the post-genomics era, the field of proteomics is being actively investigated for the clinical potential of proteins and peptides as a rich source of biological markers. As tumor cells transition from single-layer structures to multilayer masses, a unique set of proteins associated with angiogenesis, extracellular matrix, apoptosis, cell growth, and invasion are produced and shed into the circulation. Advances in mass spectrometry (MS) have enabled the discovery and identification of relevant proteins from complex samples such as serum, plasma, and other bodily fluids to provide non-invasive approaches for reliable early detection strategies.
Approach: The most challenging technical hurdle obstructing the discovery of new protein biomarker candidates is the ability to acquire access to the most clinically relevant circulating proteome in the blood. Over 90% of the blood proteins are highly abundant and characteristically possess relatively high molecular weights, such as albumin (67 kDa) and Immunoglobulins G (50 kDa). Since MS modalities such as matrix-assisted laser desorption/ionization (MALDI) normalize peak intensities to the most intense peak, high abundant proteins mask the presence of low abundant proteins. Our group has developed nanoporous silica chips (NSCs) that utilize nanoscale pores to discriminate the capture of high molecular weight proteins/peptides from complex samples such as serum and plasma. Low molecular weight (LMW) proteins are selectively captured within pores of the NSCs, and through subsequent washing steps, the high molecular weight proteome is removed. The captured LMW proteome can then be eluted from the nanopores and transferred to a MALDI plate for analysis. To enhance the selectivity of specific classes of proteins/peptides, pore sizes can be tightly engineered and controlled to very specific pore diameters, utilizing state-of-the-art silicon fabrication techniques borrowed from the semiconductor industry. In addition to controlling pore size and geometry, our laboratory is capable of modifying the surfaces of nanoporous thin films through chemistry and dopants to offer NSCs with additional sophistication for enhanced selectivity. The serum and/or plasma samples are processed utilizing the NSC technology, analyzed via MALDI, and clinically relevant peaks have been selected from MS profiles. These unknown proteins/peptides can be sequenced and identified through other MS modalities, such as liquid chromatography-mass spectrometry (LC-MS). Once a protein biomarker has been identified, this information can be used to create new early detection strategies and therapeutics.
|Sample fractionation using mesoporous silica chips. The schematic shows the four primary steps in sample processing, which results in the removal of high molecular, high abundant proteins and enrichment of low abundant, low molecular weight proteins. The elution sample is then analyzed via mass spectroscopy, and the data can be subsequently mined and analyzed.|
We recently expended the application of NSCs in the detection and quantification of target peptides from human liquid biopsies. By precisely tailoring the size, structure and surface charge of nanopores, we integrated the size-exclusion mechanism of nanoporous silica (NPS) films and the electrostatic interactions between the cationic biomarker and the negatively charged silica surface. The sensitivity and reproducibility of the platform were validated to test hepcdin, a small peptide hormone recognized as a biomarker for iron-related diseases. To promote this new approach to clinical practices, we further applied it to successfully assay the hepcidin levels in human body fluids provided by healthy volunteers and patients.
|Hepcidin detection by MALDI-TOF MS with and without enrichment on NPS thin films. (a) Mass spectrum of serum without fractionation. (b) and (c) Mass spectrums of serum and urine samples after selective enrichment.|
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