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.

Critical assessment of accelerating trypsination methods.

In LC-MS based proteomics, several accelerating trypsination methods have been introduced in order to speed up the protein digestion, which is often considered a bottleneck. Traditionally and most commonly, due to sample heterogeneity, overnight digestion at 37 °C is performed in order to digest both easily and more resistant proteins. High efficiency protein identification is important in proteomics, hours with LC-MS/MS analysis is needless if the majority of the proteins are not digested. Based on preliminary experiments utilizing some of the suggested accelerating methods, the question of whether accelerating digestion methods really provide the same protein identification efficiency as the overnight digestion was asked. In the present study we have evaluated four different accelerating trypsination methods (infrared (IR) and microwave assisted, solvent aided and immobilized trypsination). The methods were compared with conventional digestion at 37 °C in the same time range using a four protein mixture. Sequence coverage and peak area of intact proteins were used for the comparison. The accelerating methods were able to digest the proteins, but none of the methods appeared to be more efficient than the conventional digestion method at 37 °C. The conventional method at 37 °C is easy to perform using commercially available instrumentation and appears to be the digestion method to use. The digestion time in targeted proteomics can be optimized for each protein, while in comprehensive proteomics the digestion time should be extended due to sample heterogeneity and influence of other proteins present. Recommendations regarding optimizing and evaluating the tryptic digestion for both targeted and comprehensive proteomics are given, and a digestion method suitable as the first method for newcomers in comprehensive proteomics is suggested.

Subclinical effects of saxitoxin and domoic acid on aggressive behaviour and monoaminergic turnover in rainbow trout (Oncorhynchus mykiss).

The algal produced neurotoxins saxitoxin and domoic acid may have serious effects on marine life and can be responsible for the intoxication of for instance sea mammals, sea birds and fish. Given that farmed fish cannot escape algal blooms, they may be more susceptible to intoxication than wild stocks. In the present study, subclinical effects of saxitoxin and domoic on aggressive behaviour and monoaminergic systems in the brain of the rainbow trout (Oncorhynchus mykiss) were investigated. The resident-intruder test was used to measure aggression where only the resident fish were subjected to the toxins and analysed for monoamines and their metabolites. The resident-intruder test was carried out on two consecutive days. On day one basal aggression was measured in the four groups. On day two three of the groups were injected with subclinical doses of one of the following: saxitoxin (1.752 microg/kg bw), domoic (0.75 mg/kg bw) or 0.9% saline solution. This was performed 30 min prior to the aggression test. Handling stress and injection affected aggressive behaviour, cortisol and the serotonergic system in telencephalic brain regions. Cortisol levels were elevated in all of the injected groups when compared to the control group. An increase in serotonergic turnover was evident when all injected groups were pooled and compared to the control group. All together this suggests that the handling stress in connection with the injection was similar in all of the three injected groups. In contrast to both the undisturbed control group and the toxin-injected groups, the saline-injected group displayed a reduction in aggressive behaviour which was evident in increased attack latency. Furthermore the domoic injected group displayed more aggressive attacks towards their conspecifics than the saline-injected group. Consequently the two toxins appear to mask the stress induced alteration in aggressive behaviour. Monoamine levels and monoaminergic turnover could not be demonstrated to be directly affected by the two toxins at the given doses in the investigated brain regions (dorsal and ventral parts of telencephalon, optic tectum, locus coeruleus, raphe nucleus, molecular and granular layer of cerebellum). This could indicate that the toxins mediate aggressive behaviour either through other systems than the monoaminergic systems, such as neuroactive amino acids, or that the mediation occurs in other brain regions.