Unknown

Dataset Information

0

Open tubular lab-on-column/mass spectrometry for targeted proteomics of nanogram sample amounts.


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.

SUBMITTER: Hustoft HK 

PROVIDER: S-EPMC4164520 | BioStudies | 2014-01-01

REPOSITORIES: biostudies

Similar Datasets

1000-01-01 | S-EPMC3863811 | BioStudies
2007-01-01 | S-EPMC2117336 | BioStudies
1000-01-01 | S-EPMC5126632 | BioStudies
2019-01-01 | S-EPMC6702421 | BioStudies
2014-01-01 | S-EPMC3959646 | BioStudies
2017-01-01 | S-EPMC5497570 | BioStudies
2011-01-01 | S-EPMC3073582 | BioStudies
2016-01-01 | S-EPMC4824869 | BioStudies
2012-01-01 | S-EPMC3424325 | BioStudies
2020-01-01 | S-EPMC7341532 | BioStudies