Methane inhibition by Asparagopsis taxiformis with rumen fluid collected from ventral and central location – a pilot study

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

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

Integrated enzyme reactor and high resolving chromatography in ‘‘sub-chip’’ dimensions for sensitive protein mass spectrometry

Reliable, sensitive and automatable analytical methodology is of great value in e.g. cancer diagnostics. In this context, an on-line system for enzymatic cleavage of proteins, subsequent peptide separation by liquid chromatography (LC) with mass spectrometric detection has been developed using “sub-chip” columns (10–20 µm inner diameter, ID). The system could detect attomole amounts of isolated cancer biomarker progastrin-releasing peptide (ProGRP), in a more automatable fashion compared to previous methods. The workflow combines protein digestion using an 20 µm ID immobilized trypsin reactor with a polymeric layer of 2-hydroxyethyl methacrylate-vinyl azlactone (HEMA-VDM), desalting on a polystyrene-divinylbenzene (PS-DVB) monolithic trap column, and subsequent separation of resulting peptides on a 10 µm ID (PS-DVB) porous layer open tubular (PLOT) column. The high resolution of the PLOT columns was maintained in the on-line system, resulting in narrow chromatographic peaks of 3–5 seconds. The trypsin reactors provided repeatable performance and were compatible with long-term storage

Graphical overview of the OTER-PLOT nanoproteomic platform.

<p>Graphical overview of the OTER-PLOT nanoproteomic platform.</p

Total ion chromatogram (TIC) of a fully automated digested, separated and detected HCT15 lysate, leading to the identification of ∼1500 proteins (data recorded from gradient start).

<p>OTER volume was approximately 1.2 µL.</p

SEM image of the OTER (20 µm ID) (the immobilized enzymes are too small to be visualized in the image).

<p>The polymeric layer was ∼0.4 µm (dry).</p

Carry-over of the highest abundant peptide ions in a blank following injection of a 10 protein standard mix.

<p>Upper chromatogram: TIC of the standard protein mixture, lower chromatograms; zoom-in of three of the highest abundant ions in the standard protein mix compared to the same ions in the blank (not able to extract). OTER volume was approximately 1.2 µL.</p

A. EIC of identified peptide TSVQPSHLFIQDPTMPPHPAPNPLTQLEEAR corresponding to Axin1 in standard mixture, HCT15 cell protein extract digested off-line and on-line.

<p>B. EIC of identified peptide HETGSHDAER corresponding to APC in protein extracts from HCT15 cell line and HCT15 xenograft, respectively. OTER volume was approximately 1.2 µL.</p