Abstract
A novel open tubular nanoproteomic platform featuring accelerated on-line protein digestion and high-resolution nano liquid chromatography mass spectrometry (LC-MS) has been developed. The platform features very narrow open tubular columns, and is hence particularly suited for limited sample amounts. For enzymatic digestion of proteins, samples are passed through a 20 µm inner diameter (ID) trypsin + endoproteinase Lys-C immobilized open tubular enzyme reactor (OTER). Resulting peptides are subsequently trapped on a monolithic pre-column and transferred on-line to a 10 µm ID porous layer open tubular (PLOT) liquid chromatography LC separation column. Wnt/ß-catenein signaling pathway (Wnt-pathway) proteins of potentially diagnostic value were digested+detected in targeted-MS/MS mode in small cell samples and tumor tissues within 120 minutes. For example, a potential biomarker Axin1 was identifiable in just 10 ng of sample (protein extract of ∼1,000 HCT15 colon cancer cells). In comprehensive mode, the current OTER-PLOT set-up could be used to identify approximately 1500 proteins in HCT15 cells using a relatively short digestion+detection cycle (240 minutes), outperforming previously reported on-line digestion/separation systems. The platform is fully automated utilizing common commercial instrumentation and parts, while the reactor and columns are simple to produce and have low carry-over. These initial results point to automated solutions for fast and very sensitive MS based proteomics, especially for samples of limited size.
Highlights
As cancer medicine evolves towards personalized treatment, the need for high throughput, selective and sensitive methods for monitoring key diagnostic-value biomolecules is increasing [1,2,3]
Much diagnosis-relevant sample measurements are expected to be performed at the gene/mRNA level, and several high-throughput genetic testing methods have been developed for e.g. breast, colon, prostate and colorectal cancer [4,5,6,7]
The rate-limiting step of the enzymatic cleaving of proteins is the use of relatively slow in-solution procedures. Another goal is to develop procedures that accelerate the enzyme reaction step: one approach is the use of immobilized enzyme reactors (IMERs), as they have several advantages compared to standard in-solution/in-gel approaches, such as larger enzyme to substrate ratio, higher digestion efficiency, long-term stability and reusability [29,30]. For improving both LC separation and accelerate enzymatic digestion, we have recently demonstrated the proof of principle of using a ‘‘lab-on-a column’’ system with open tubular columns
Summary
As cancer medicine evolves towards personalized treatment, the need for high throughput, selective and sensitive methods for monitoring key diagnostic-value biomolecules is increasing [1,2,3]. In this regard, much diagnosis-relevant sample measurements are expected to be performed at the gene/mRNA level, and several high-throughput genetic testing methods have been developed for e.g. breast, colon, prostate and colorectal cancer [4,5,6,7]. MS based approaches can provide additional information with higher throughput, selectivity or sensitivity compared to e.g. WB, ELISA, DNA and mRNA sequencing [11,12,13,14]. A goal in the LC-MS based proteomics is to develop novel LC columns/materials that provide improved separation, i.e. high peak capacity; contemporary approaches include development of monolithic materials (silica and organic [25,26]), ultra high performance LC (UHPLC) [27] and fused core particle materials [28]
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