Proteome Sciences Share Discussion Threads

[colinhy] 21/3/2021 21:57

Proteome Sciences TMT technology features in the following research published March 15 2021.

I note that the Oxford AstraZeneca Covid19 vaccine creators Sarah Gilbert and Susan Morris at the Oxford univerisity Jenner Institute are among the authors.


"SARS-CoV-2 vaccine ChAdOx1 nCoV-19 infection of human cell lines reveals low levels of viral backbone gene transcription alongside very high levels of SARS-CoV-2 S glycoprotein gene transcription."


Background
ChAdOx1 nCoV-19 is a recombinant adenovirus vaccine against SARS-CoV-2 that has passed phase III clinical trials and is now in use across the globe. Although replication-defective in normal cells, 28 kbp of adenovirus genes is delivered to the cell nucleus alongside the SARS-CoV-2 S glycoprotein gene.

Methods
We used direct RNA sequencing to analyse transcript expression from the ChAdOx1 nCoV-19 genome in human MRC-5 and A549 cell lines that are non-permissive for vector replication alongside the replication permissive cell line, HEK293. In addition, we used quantitative proteomics to study over time the proteome and phosphoproteome of A549 and MRC5 cells infected with the ChAdOx1 nCoV-19 vaccine.

Total and phosphoproteome analysis
Aliquots of 100 μg of each sample were digested with trypsin (2.5 μg trypsin per 100 μg protein; 37 °C, overnight) and labelled with Tandem Mass Tag (TMT) ten plex reagents according to the manufacturer’s protocol (Thermo Fisher Scientific, Loughborough, LE11 5RG, UK), and the labelled samples pooled according to cell line.

For the phosphoproteome analysis, the remainder of the TMT-labelled pooled sample was also desalted using a SepPak cartridge (Waters, Milford, MA, USA). Eluate from the SepPak cartridge was evaporated to dryness and subjected to TiO2-based phosphopeptide enrichment according to the manufacturer’s instructions (Pierce).

Conclusions
Our analyses provide valuable insight into the transcriptomic and proteomic repertoire of an important vaccine, providing confirmation that the vaccine vector’s transcriptome is essentially as intended in these cell lines. However, care should be taken when extrapolating the results of the transcriptomic analysis using single cell types to the complex multicellular differentiated cell-rich environment these types of vaccines encounter when they are administered (by whatever route). Recently, an in-depth multi-omic analysis of the herpes virus genome revealed that an oncolytic herpes virus licenced in 2015 under the name Imlygic is in fact deleted for an additional third gene rather than the two intended because of a previously unknown ORF present in the deleted region [49]. Whilst there is no suggestion that this has been a problem, it highlights the importance of utilising state-of-the-art and unbiased approaches to survey genetically modified viruses intended for clinical use. Finally, we argue that this kind of analysis is relatively straightforward and should be routinely incorporated into the early stages of future viral vector evaluation pipelines to allow a robust understanding of the transcriptomic potential of engineered viral vectors.




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for info
How the ‘Oxford’ Covid-19 vaccine became the ‘AstraZeneca’ Covid-19 vaccine

[tom barnaby] 21/3/2021 21:24

BOL!

Look at the rubbish the avatar has been cutting and pasting for years and save yourself the bother of reading the reply.

[dk37] 21/3/2021 21:03

colinhy

Thank you for your response. Can i assume, given your post , that you believe this new development may be of some significance for prm over the near term.

[tom barnaby] 21/3/2021 17:18

58650 re 58648 see 58649

[colinhy] 21/3/2021 17:10

dk37
Re your Post #58648

TMT quantitation of plasma protein analysis is one of a number of methodologies used in proteomic plasma analysis and is therefore not a fundamental component of all methods of plasma protein analysis.

[dk37] 21/3/2021 16:01

colinhy

Many thanks for the above.

I have seen many studies linking TMT with plasma analysis. Can you tell me if TMT is a fundamental component of these new, and very exciting, plasma developments?

if so it indicates a new and possible significant new growth stream.

[colinhy] 21/3/2021 12:03

Plasma Proteomics Sees Substantial Advances in Depth of Coverage
Mar 18, 2021 | Adam Bonislawski


NEW YORK – Several proteomics firms have recently highlighted new mass spectrometry-based offerings that they say will significantly add to the number of proteins quantified in a typical plasma proteomic experiment.

The moves indicate that proteomic technologies are making substantial progress in plasma analysis, an area that has long been among the most challenging in the field.

This month, Swiss proteomics firm Biognosys said that it expects by the end of the year to begin offering a new discovery proteomics workflow that will allow it to quantify around 2,700 proteins in a typical plasma study, and that it expects to manage around 3,300 proteins in large-scale discovery studies by the end of 2022.

Also this month, Qing Wang, CEO of Baltimore, Maryland-based multi-omics company Complete Omics, said that the firm planned by the end of the year to launch a targeted mass spec assay that will quantify 4,550 proteins in plasma.

And at the US Human Proteome Organization's annual meeting this month, researchers from Bruker and proteomics firm Seer showed that a workflow combining Seer's Proteograph system with Bruker's timsTOF Pro mass spectrometer was able to quantify more than 1,700 proteins in plasma. That followed a study published by Seer last summer in Nature Communications in which the company used its Proteograph system to identify roughly 2,000 proteins in an analysis of 141 plasma samples.

Existing mass spec-based plasma protein assays typically measure somewhere in the range of 300 to 500 proteins in undepleted plasma and between 500 and 1,000 proteins in depleted plasma, meaning the Seer assay and the proposed Biognosys and Complete Omics workflows mark a significant jump in the depth of plasma proteomic analyses.

In the case of Complete Omics, the company will achieve this depth by using extensive sample fractionation, which will set it apart from most recent plasma proteomics efforts where throughput has become a major priority. According to Wang, running the complete assay will take around 10 hours per sample, which makes it poorly suited to the sort of large-scale experiments many researchers are interested in pursuing. Wang said he expected that for most projects the company would run a more targeted subset of the panel determined by the specific clinical questions they were trying to answer.

At Biognosys, the company's deep plasma proteomic workflow will be compatible with large-scale experiments as they will be capable of analyzing hundreds of samples in "a few weeks," said Lukas Reiter, Biognosys' chief technology officer.

Reiter said the improvements in depth were due to refinements across all portions of the mass spec sample prep and analysis process that had expanded the dynamic range covered by such experiments. This expansion of dynamic range has allowed the company to move into an abundance range where many more proteins are present.

"If you are in an abundance range were there are simply a lot of proteins, then any increase in your dynamic range will have a big effect on the amount of proteins you gain," he said.

Reiter noted that the field previously saw a similar phenomenon with experiments in cell lines.

"There was a time in the past when increasing dynamic range led to huge gains in the number of proteins [identified in cell lines]," he said, adding that the field was now on the cusp of a similar development in plasma.

Reiter said that when Biognosys started offering plasma proteomic assays it was quantifying proteins across a dynamic range of four to five orders of magnitude. Its new assay will measure across a dynamic range of six to seven orders of magnitude, he said.

He attributed the advance to a number of technological improvements, including advances in protein depletion workflows as well as liquid chromatography and the mass spec systems themselves.

"There have been big steps in chromatography over the last 10 years, and there are more coming," he said. "And then mass spectrometers have become much faster, which essentially means increasing your dynamic range."

The additional dimensions of separation provided by ion mobility systems like Bruker's trapped ion mobility (TIMS) and Thermo Fisher Scientific's FAIMS have also contributed to the improved depth of coverage, Reiter said.

"The major contributor here is the fact that all of these things can be combined and they all add up to an increase in dynamic range such that you are coming into a range where there are just more proteins around," he said.

Addressing the dynamic range challenges involved in analyzing the plasma proteome is also at the heart of Seer's Proteograph platform. The Redwood City, California-based company uses nanoparticle-based enrichment of proteins in samples like human plasma to enable deeper coverage in proteomic discovery experiments. The platform is based on the observation that when incubated in a biological sample, nanoparticles collect proteins, which form a "corona." This enables nanoparticles to serve as an enrichment tool, allowing researchers to pull proteins out of a sample, which they can then identify and quantify using technologies like mass spec or other detectors.

The company currently offers a panel of five nanoparticles with which it and its collaborators have quantified in the range of 1,500 to 2,000 proteins in plasma experiments.

The lab of Mark Flory, a researcher at the Oregon Health and Science University's Knight Cancer Institute, which is one of the first early-access sites working with Seer's technology, installed the Proteograph system at the end of 2020 and has been doing pilot experiments with the platform in order to determine its effectiveness before using it in large sample cohorts held by external collaborators.

Thus far, Flory, who was a co-author on the Bruker and Seer poster at US HUPO, said he has been impressed with the results.

"We did some initial comparison studies comparing it to traditional methods that have been used for discovery in plasma using fractionation and/or depletion, and Seer really outperformed those," he said. "It did better and with less labor, less time for preparation, and less time for data acquisition."

The system has also shown good reproducibility, Flory said, noting that this was a major concern of his initially and likely a major concern of others in the field.

"We were somewhat skeptical," he said. "We've seen a lot of platforms come through the proteomics field, but this one actually worked as advertised."

Flory said that based on his experience with the system so far, he anticipates using it for all the plasma samples his lab runs.

"There's just really no other alternative that allows you to do a scaled study for discovery or even validation of proteins in the mixtures easily," he said.

Like many researchers doing plasma proteomics work, Flory is keen to avoid protein depletion steps. While depletion improves depth of coverage by removing the most high-abundance proteins from a sample, it can be time-consuming, expensive, and cause issues with reproducibility.

"One thing we have always been concerned about is that as you deplete high-abundance proteins you may non-specifically deplete some of the [proteins] you are actually going after," he said. "There's not 100 percent specificity with those depletion methods, so that is something we have really shied away from, even though it does help" depth of coverage.

Reiter said, though, that Biognosys found depletion useful, noting that protocols and reproducibility and efficiency have improved.

Jochen Schwenk, director of translational plasma profiling at the Science for Life Laboratory at Sweden's Royal Institute of Technology, said that he believed announcements like that from Biognosys were not unexpected given the progress that has been made in plasma proteomics in recent years, though he noted that "the real benefit will come once we are able to routinely detect all of these [proteins] in all samples."

Philipp Geyer, chief scientific officer and co-founder of German plasma proteomics firm OmicEra Diagnostics, similarly noted that the numbers advertised by Biognosys reflected not the total number of proteins the company expected to quantify per sample but, rather, the number it expected to quantify over the course of an entire study.

"If you are going to do a complete study where you measure hundreds of samples or even thousands of samples, then of course you will find in one sample a protein that you haven't seen before in another sample," he said. "That is what we usually see, that if you go to larger and larger studies, you find more and more proteins."

He added that natural variation between samples could also add to the total number of proteins quantified in a study.

In the study published by Seer scientists this summer in Nature Communications, the researchers found in their analysis of 141 plasma samples that of the more than 2,000 proteins identified, 1,992 were quantified in at least 25 percent of all samples, suggesting that the per-sample number was somewhat lower than that.

Geyer said that in a typical plasma protein study, researchers might expect to quantify more than 1,000 proteins across all the samples measured in the study. Given that, the 2,700 proteins per study Biognosys aims to hit by the end of the year would still be a substantial advance.

Geyer who led several large-scale plasma proteomics experiments while a researcher in the lab of Max Planck Institute of Biochemistry researcher Matthias Mann, said that while he believed that "sooner or later" improved plasma proteomic coverage "will happen for sure," he was less optimistic that the field was currently on the cusp of seeing a significant increase in depth of coverage.

He said that his impression, based on his reading of the field, was that capabilities were still in the range of around 300 to 500 proteins per sample in undepleted plasma and somewhat more than that in depleted plasma.

That said, a jump in profiling depth "would be very nice to see," he said.



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For info

[tom barnaby] 20/3/2021 22:26

Aye, about 17 years ago.

[mashman] 20/3/2021 22:18

Charts gone negative. Time to sell

[1xtrader] 20/3/2021 22:03

What is it then lol

[tom barnaby] 20/3/2021 21:57

I certainly could.

[1xtrader] 20/3/2021 21:50

As Michael Douglas says in Wall Street "tell me something I don't know"Tick tock

[tom barnaby] 20/3/2021 21:46

No idea, amateur hour at the Lancet is best left to those that think they know better, but inevitably don’t.

The sales figures over the last decade plus suggest that those that do know better may not think so.

However, things may be about to change, if certain information that some avatars may or may not be in possession of comes through.

[1xtrader] 20/3/2021 21:30

Ok could tmt pro be a tool to help find a cure for certain Diseases

[1xtrader] 20/3/2021 21:15

Proteome sciences has made technological break through that will cure diseases is this a true statement

[tom barnaby] 20/3/2021 21:08

Not at al 1xtrader.

There is no other share on the LSE that has ever offered as much entertainment as this. What’s more, it’s never cost me a cent.

[1xtrader] 20/3/2021 19:56

10 million is peanuts

[tom barnaby] 20/3/2021 15:18

Oh and just in case it does somehow manage to get off the worlds biggest roundabout and turn things around, he can convert that debt into shares for next to F all, thus diluting LTHs heavily underwater holdings significantly.

[tom barnaby] 20/3/2021 15:14

£10m or so that is repayable on demand to the CEO for starters. A debt that has a full charge against the assets if they can’t stump up the readies.

[1xtrader] 20/3/2021 14:53

What is the debt I thought the account were reasonable

[tom barnaby] 20/3/2021 14:31

Sounds good 1xtrader, which stock are you on about?

It would make a nice change from avatars flogging this debt ridden husk that has failed in ever single one of its poorly thought through ventures over more than 2 decades.