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Triple quad TQ-S

Triple quad TQ-S | Mass Spectrometry Geekery | Scoop.it

Unlike the synapt, I personally, do get my hands on the new TQ-S. It lives in Lionel's lab at JIC. I want to use our standard mix of proteins (the quantified UPS2 standard) to see how sensitive I can get it. The objective is to match the demo data.

 

As this instrument is only being used intermittently the source is general kept closed (arrow down in picture) this nearly caught me out of my first day back on the instrument  - these basic gaffes could kill a lot of time! The arrow should be horizonal so that ions (and air) can enter. The little source cone is off here, this is one of the easiest to clean, just a twist pull. Nice :)

 

A few other little glitches kept my first days interesting; the autosampler had been dropped out of instrument config - that fooled me for a while; getting the blunt-end nano spray fitting together is always fun - without a dead-end stop the two silicon capillary ends grind against each other as they slide in the junction. When I found the correct stop and flushed out the broken glass things went better :)

 

After a couple of days leaning and fixing the set-up I have a reasonable method for the UPS2 standard mix and - yes! - recreated the demo data test as a bench mark to the orbitrap performance. I am happy to say that, for most peptides, this compares favourably and I can reliably observe the lower abundabce peptides that are not often seen on the orbi.

 

[edit 25th April '12. Darn - the arrow-error caught me out again *headdesk* Thank you Lionel for spotting this immediately when I complained about getting no signal.)

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Advances in characterizing ubiquitylation sites by mass spectrometry

Advances in characterizing  ubiquitylation sites by mass spectrometry | Mass Spectrometry Geekery | Scoop.it
alex's insight:

This is a great current review. I have a couple of minor quibbles in Figure 1. Firstly - I believe that only HECT E3 ligases become ubiquitinated themselves as part of their reaction mechanism and that RING E3 ligases do not.  Secondly, I love that in Fig 1b a 'typical MS/MS' experiment involves an Orbitrap! Other types of mass spectrometer exist and could be used just/nearly* as well.

 

On a more serious note they discuss the concern over alkylating agent artefacts that can produce the same mass shift as the GlyGly residues left after tryptic digestion of ubiquitinated proteins. They clearly favour a QExactive which goes someway to explaining Figure 1b. Overall it’s a great read and a nice summary of recent ubiquitination papers.

 

One last question they did not address though – is it ‘ubiquitination or ubiquitylation’? Even Wiki is undecided http://en.wikipedia.org/wiki/Ubiquitin#Ubiquitination_.28ubiquitylation.29

 

*delete according to taste

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Comment on "Protein Sequences from Mastodon and Tyrannosaurus rex Revealed by Mass Spectrometry"

alex's insight:

It is rare that a technical commentary is witty and quite as scathing as this comment by Pavek Pevzner et al. Ok so it's a little old now but I was checking out his more recent publications and remembered this gem.

 

It starts "Imagine a monkey typing random keys on a typewriter and let us assume that the monkey is given 100,000 attempts to generate six-letter words. One would be surprised if the monkey typed a six-letter word from Webster's dictionary on the first attempt; indeed, the probability of this is rather low. However, nobody would be surprised if some of the 100,000 words turned out to be correctly spelled English words. "

 

Abstract:

Asara et al. (Reports, 13 April 2007, p. 280) reported sequencing of Tyrannosaurus rex proteins and used them to establish the evolutionary relationships between birds and dinosaurs. We argue that the reported T. rex peptides may represent statistical artifacts and call for complete data release to enable experimental and computational verification of their findings.

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Nature Method of the Year 2012 : Targeted Proteomics

New method and tool developments are helping to bring targeted proteome analysis technologies to a broader array of biologists.
alex's insight:

Good to see this approach getting the recognition it deserves, and watch other for alternative methods like SWATH too! So this is a nice primer to pass around to convince people that you need a new mass spec for the new year!

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Rajesh Pujari's curator insight, March 18, 2013 12:18 PM

Mass spec reaching a new height

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Mass spectral molecular networking of living microbial colonies

Mass spectral molecular networking of living microbial colonies | Mass Spectrometry Geekery | Scoop.it

 

Nice work, I love the liquid bridge concept!

 

Jeramie Watrousa,b,c, Patrick Roachd,1, Theodore Alexandrovc,e, Brandi S. Heathd, Jane Y. Yanga,b,c, Roland D. Kerstena,b,f, Menno van der Voortg, Kit Poglianoh, Harald Grossi, Jos M. Raaijmakersg, Bradley S. Moorec,f, Julia Laskind,2, Nuno Bandeirac,j,k,2, and Pieter C. Dorresteina,b,c,f,2

 

Abstract

Integrating the governing chemistry with the genomics and phenotypes of microbial colonies has been a “holy grail” in microbiology. This work describes a highly sensitive, broadly applicable, and cost-effective approach that allows metabolic profiling of live microbial colonies directly from a Petri dish without any sample preparation. Nanospray desorption electrospray ionization mass spectrometry (MS), combined with alignment of MS data and molecular networking, enabled monitoring of metabolite production from live microbial colonies from diverse bacterial genera, including Bacillus subtilis, Streptomyces coelicolor, Mycobacterium smegmatis, and Pseudomonas aeruginosa. This work demonstrates that, by using these tools to visualize small molecular changes within bacterial interactions, insights can be gained into bacterial developmental processes as a result of the improved organization of MS/MS data. To validate this experimental platform, metabolic profiling was performed on Pseudomonas sp. SH-C52, which protects sugar beet plants from infections by specific soil-borne fungi [R. Mendes et al. (2011) Science 332:1097–1100]. The antifungal effect of strain SH-C52 was attributed to thanamycin, a predicted lipopeptide encoded by a nonribosomal peptide synthetase gene cluster. Our technology, in combination with our recently developed peptidogenomics strategy, enabled the detection and partial characterization of thanamycin and showed that it is a monochlorinated lipopeptide that belongs to the syringomycin family of antifungal agents. In conclusion, the platform presented here provides a significant advancement in our ability to understand the spatiotemporal dynamics of metabolite production in live microbial colonies and communities.

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Label-Free Quantitation of Protein Modifications by Pseudo Selected Reaction Monitoring with Internal Reference Peptides - Journal of Proteome Research (ACS Publications)

Label-Free Quantitation of Protein Modifications by Pseudo Selected Reaction Monitoring with Internal Reference Peptides - Journal of Proteome Research (ACS Publications) | Mass Spectrometry Geekery | Scoop.it

If you are interested in triple quad type quantitation but have an ion trap – or simply want to make the most of your existing ion trap data – then check out the developments in Skyline described in this paper or with the great tutorials available at https://brendanx-uw1.gs.washington.edu/labkey/project/home/software/Skyline/begin.view

 

 

Abstract

Liquid chromatography tandem mass spectrometry (LC–MS/MS) based methods provide powerful tools for the quantitative analysis of modified proteins. We have developed a label-free approach using internal reference peptides (IRP) from the target protein for signal normalization without the need for isotope labeling. Ion-trap mass spectrometry and pseudo-selected reaction monitoring (pSRM) were used to acquire full MS/MS and MS3 spectra from target peptides. Skyline, a widely used software for SRM experiments, was used for chromatographic ion extraction. Phosphopeptides spiked into a BSA background yielded concentration response curves with high correlation coefficients (typically >0.9) and low coefficients of variation (≤15%) over a 200-fold concentration range. Stable isotope dilution (SID) and IRP methods were compared for quantitation of six site-specific phosphorylations in the epidermal growth factor receptor (EGFR) in epidermal growth factor-stimulated A431 cells with or without the addition of EGFR inhibitors cetuximab and gefitinib. Equivalent responses were observed with both IRP and SID methods, although analyses using the IRP method typically had higher median CVs (22–31%) than SID (10–20%). Analyses using both methods were consistent with immunoblot using site-selective antibodies. The ease of implementation and the suitability for targeted quantitative comparisons make this method suitable for broad application in protein biochemistry.

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Targeted Proteomic Quantification on Quadrupole-Orbitrap Mass Spectrometer

And the next item on my wish-list is....probably one of these

 

 

Sebastien Gallien,Elodie Duriez,Catharina Crone2,Markus Kellmann,Thomas Moehring andBruno Domon

 

Abstract

There is an immediate need for improved methods to systematically and precisely quantify large sets of peptides in complex biological samples. While to date protein quantification in biological samples is routinely performed on triple quadrupole instruments operated in selected reaction monitoring mode (SRM), two major challenges remain. Firstly, the number of peptides to be included in one survey experiment needs to be increased to routinely reach several hundreds, and secondly, the degree of selectivity should be improved to reliably discriminate the targeted analytes from background interferences. High resolution and accurate mass (HR/AM) analysis on the recently developed Q-Exactive mass spectrometer can potentially address these issues. This instrument presents a unique configuration: it is constituted of the orbitrap mass analyzer equipped with a quadrupole mass filter as front-end for precursor ion mass selection. This configuration enables new quantitative methods based on HR/AM measurements, including targeted analysis in MS mode (single ion monitoring, SIM) and in MS/MS mode (parallel reaction monitoring, PRM). While the ability of the quadrupole to select a restricted m/z range allows overcoming the dynamic range limitations associated with trapping devices, the MS/MS mode provides an additional stage of selectivity. When applied to targeted protein quantification in urine samples and benchmarked with the reference SRM technique, the quadrupole-orbitrap instrument exhibits similar or better performances in terms of selectivity, dynamic range, and sensitivity. These high performances are further enhanced by leveraging the multiplexing capability of the instrument to design novel acquisition methods and apply them to large targeted proteomic studies for the first time, as demonstrated on 770 tryptic yeast peptides analyzed in one 60 minute experiment. The increased quality of quadrupole-orbitrap data has the potential to improve existing protein quantification methods in complex samples and to cope with the pressing demand of systems biology or biomarker evaluation studies.

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mMass - Open Source Mass Spectrometry Tool

mMass - Open Source Mass Spectrometry Tool | Mass Spectrometry Geekery | Scoop.it
mMass presents an open source multi-platform software for precise mass spectrometric data analysis.

 

What a great program this is. I have reached the point in a hunt for a crosslinked peptide where I need to quickly compare fragmentation spectra. This really does the job and has lots of other nice features (that will keep me leaning for a while longer). Thanks to Martin Strohalm (http://ms.biomed.cas.cz/staff-strohalm.php)

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Orbitrap Mass Spectrometer arrived!

Orbitrap Mass Spectrometer arrived! | Mass Spectrometry Geekery | Scoop.it

It is great to see other people as excited by a shiney new mass spec. Congratulations Magnus and Helle, I hope you have lots of fun (and excellent data). 

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Talking of Step waves...

Talking of Step waves... | Mass Spectrometry Geekery | Scoop.it

…here is ours (JIC's Xevo TQ-S) during our training a few weeks ago. We had our training on-site that allowed eight people from three institutes to dip in and out of the few lectures and mostly lab-based demonstrations provided by SC. It was great fun and very useful. We have been using the instrument for a while and have had training on the Synapt just a few months ago so certainly I feel quite confident and the training cleared us a few niggling questions. I think the same is true for the others.

 

As you can see from this blog I had started off going straight to MRM design, primarily using Skyline to design transitions from Orbitrap data. Since the training I have been exploring using ‘Product Ion Conformation’ which use a transition to initiate a fragment scan. This is very useful because fragmentation patterns of peptides could differ between the ion trap CID of the LTQ Orbitrap and the fragmentation seen in the travelling wave collision cell of the Xevo. I was expecting to see y ion formation favoured in the Xevo, while the ion trap produces both b and y ions. My initial testing of b ion MRMs seemed to confirm this but now, looking at some fragment spectra from the xevo some b ions are produced after all. I would like to get these fragment spectra into Mascot and from there into Scaffold – my favourite analytical tools, but so far I have been a bit stuck on the export of the fragment spectra in a suitable format. This is so frequently the case with MS data and I would welcome any tips on how to easily export all the fragment ion spectra from such PIC scans generated from an LC run. My current options seem to be an involved manual clicking process or a batch export to excel and then I could write a Perl script to make mgfs from there. It’s almost a shame that I have other plans for the weekend as I quite enjoy messing around with a little Perl challenge (not that it would take all weekend; I’m not that weak!)

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Kalibr's comment, April 11, 2012 11:26 AM
I'm curious to see what you get out of the ‘Product Ion Conformation’ scans, especially at concentrations closer to the limits of sensitivity of the triple quad's MRM. I understand you bought a QQQ for going straight to MRM design but didn't you ever consider it would be really nice if you could confirm by MS/MS spectra as easily as you could trigger it by MRM? I'm taking for grated you decided that the QQQ was the way to go. To my knowledge the only QQQ mass spec that can acquire MS/MS spectra with high sensitivity is a QTRAP something that's innate to that system. The concepts are all well published and if you enjoy technology, I recommend reading the James Hager paper and patents (inventor). I really admire the new development of the accelerator trap that further improves the original concept of the axial extraction of the LIT.If you read on the ScanWave, you realize it's an interesting concept for a world with no such thing as a QTRAP. The collision cell application for enriching the Q3 scan is interesting but you soon realize if it worked properly, why would you have the Q3 scanning anyway?! On the other hand, QQQ wise it's a solid high sensitivity system and you should do fine for what you intend to do.
alex's comment, April 12, 2012 5:43 AM
Hi Kalibr,

I will post more about PICs soon (and indeed submit a real paper for review). So far the fragmentation spectra themselves look good but I am having problems getting the peaks lists out of the MassLynx file and into something that I am more comfortable with (Mascot or Scaffold for preference). I am talking to Waters about this.
Regarding the choice of TQS vs. QTrap we did indeed look closely at the 5500 as we have a QTrap 4000 on site and I am familiar with the first model of QTrap too. So I was perhaps a little biased to go that way myself. But the MS Instruments here are shared with many users and applications therefore the choice was influenced also by how easily we can switch between ordinary ESI and nanospray sources. The AB instruments have to be vented to change the source while others do not. So that was one point in many that influenced that purchase.
Essentially though I agree, it would be great to have very sensitive confirmation scans and I am not sure yet where the limit for reasonable PICs are on the TQS in comparison to something with a trap (and that for me is primarily and orbitrap xl; the 4000 I mentioned above is mostly used by a metabolite group). The fragmentations will vary with how well the peptide behaves too and while I will look at this using something like the UPS2 standard mix I will not do a systematic analysis. My projects are mostly using a few well characterised proteins, for which I have standards and lots of orbi data.
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Model Misinterpretation within Biology: Phenotypes, Statistics, Networks, and Inference | Frontiers in Plant Systems Biology

Model Misinterpretation within Biology: Phenotypes, Statistics, Networks, and Inference | Frontiers in Plant Systems Biology | Mass Spectrometry Geekery | Scoop.it

This paper raises some interesting questions in my mind. In particular regarding the validity of log 2 transformations of data as this is very popular for the analysis of 15N quantitative proteomic experiments. Our lab has started to use rank product analysis for 15N quantitation and a good commentary was made by Koziol (http://www.ncbi.nlm.nih.gov/pubmed/20093118 - and the original paper by Breitling et al is well worth a read too, also linked from above) . So how does an organism measure changes in its protein concentration (and modifications)? Do they use an absolute or log scale?

 

Model misinterpretation within biology: phenotypes, statistics, networks, and inference

Daniel J. Kliebenstein

 

Abstract

Models of myriad forms are rapidly becoming central to biology. These range from statistical models that are fundamental to the interpretation of experimental results to ordinary differential equation models that attempt to describe the results in a mechanistic format. Models will be more and more essential to biologists but this growing importance requires all model users to become more sophisticated about what is in a model and how that limits the usability of the model. This review attempts to relay the potential pitfalls that can lie within a model.

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If the antibody fails – a mass Western approach

If the antibody fails – a mass Western approach | Mass Spectrometry Geekery | Scoop.it

This paper by Lehmann et al is one of many extrolling the virtues of MRM for distinguishing between closely related proteins. I like this paper a lot and when I was contributing to the case for purchasing a new triple quad MS, this is one of the papers I would direct group leaders towards because it does not focus too much on the MS technical details but nicely illustrates the scientific case for having such an instrument. Also it uses plant material as to many research groups here and that helps sell the story.

 

So we got the new MS and now one of my first projects is to do more or less exactly this; distunguish and quantify two closely related proteins in a tryptic digest of a microsome preparation. Daunting stuff! (Daunting becase this is a complex mixture and the targets are not that abundant.) Fortunately, I have many MS spectra for one of the targets from previous work, and the group have expressed both proteins (separately) in E. coli. Many of the transitions for the second target are theoretical (I have no previous MS spectra).

 

Thus far though it looks OK - Skyline is correctly designing transitions and my method development using the expressed proteins is fine (of course I can and will collect fragmentation spectra too but it is fun to test the software). [edit: Alright, this is hard - transitions that behave perfectly with the standard (image on the left) vanish into crazy noise (on the right) with the peptides from a complex membrane digest. Not surprising I know, but I was so optimistic that I was watching peaks come off the MS. Y'know, we've all done that at times.]

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refining transitions

refining transitions | Mass Spectrometry Geekery | Scoop.it

Returning to the TQS and transition development after the break, instrument set up has been ok and Skyline continues to be nothing less than brilliant. The image shows a screenshot that combines data from the Orbitrap, TQS (and incidentally Mascot). Until you have wrestled with Xcalibur and MassLynx it is hard to explain quite how great this is - but I'll try. Xcalibur and MassLynx are products of two separate MS manufacturers, the prorgams are somewhat complicated in their own right (and understandably so) and finding a way to compare data from both is not easy. Very happily though Skyline makes this easy and I can simply get on with the job of setting up the mass spec and trying to design and choose more transitions.

 

So what we have in this image is; left long panel a list of peptides and proteins from my standard mix; top right a fragmentation spectrum from the orbitrap for a peptide; middle top the results from the TQS for that peptide; bottom a linear regression to calculate retention times of peptides. Simply wonderful! Sure there are a few glitches but at the moment I think that is my learning curve.

 

I aim to get things a bit more sensitive and establish some reasonable benchmarks, initially comparing sensitivity to what we typically see on the Orbitrap (I am using the same standard mix). Then I must turn to a real project where I already have some interesting samples waiting.

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Designing transitions: Skyline

Designing transitions: Skyline | Mass Spectrometry Geekery | Scoop.it

The strength of the triple quad is to use prior knowledge of how peptides (in my case) fragment to detect them reproducibly in complex mixtures. This is a critical and non-trivial step. I am using Skyline - a wonderful open source program developed by the MacCoss Lab - to mine our extensive collection of peptide spectra collected on our Orbitrap and develop methods for the TQS.

 

So far this is working pretty well; many transitions from the standard UPS2 mixture have behaved. Stright off the bat I am down to 500 attomoles on column. When I optimise the spray position more, change to a better column, define retention tiems and play with collision energies I think 5 attomoles is within our reach.

 

Things have gone so well that I am now looking at a 'real' protein and trying to have a quick look at some phosphopeptides before I go on holiday.

 

[edit Apil 2012 - actually 5 attomoles on column is still below my reach. 100 attomoles, yes if everything is in tip-top condition (clean but not absorbant column, clean source, stable spray, no stray polymers...oh yes and nice tight peaks - no post-column dead volume!)]

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OLIgo Mass Profiling (OLIMP) of Extracellular Polysaccharides | JoVE Video

OLIgo Mass Profiling (OLIMP) of Extracellular Polysaccharides | JoVE Video | Mass Spectrometry Geekery | Scoop.it
A rapid way is described to gain insights into the structure of polysaccharides in an extracellular matrix. The method takes advantage of the specificity of…
alex's insight:

Video is such a good way to share protocols and cover the detail that is not so obvious from a paper. JoVE is well worth browsing and contributing to!

 

Also I love that intact seedlings are just shoved into the mass spec :)

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Substrates of IAP Ubiquitin Ligases Identified with a Designed Orthogonal E3 Ligase, the NEDDylator

Substrates of IAP Ubiquitin Ligases Identified with a Designed Orthogonal E3 Ligase, the NEDDylator | Mass Spectrometry Geekery | Scoop.it
alex's insight:

A neat idea if you know your E3 ligase of interest and want to find the substrates.

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Nanomechanical mass spectrometry

Nanomechanical mass spectrometry | Mass Spectrometry Geekery | Scoop.it

I can’t help but picture a miniature steam-punk device when I hear of nanomechanical mass spectrometry. However, the reality is very cool; detection of single molecules or even single protons! Wow that could be the next big step forward in mass spectrometry. So I have pulled together a few recent papers and some public commentary for your enjoyment.

 

The Zettl Research Group at Lawrence Berkeley National Laboratory and University of California at Berkeley provide a great introduction and I’ve used their image (thanks!) http://www.physics.berkeley.edu/research/zettl/projects/nanobalance/mass.html

 

Weighing Molecules One at a Time: Physicists Create First-Ever Mechanical Device That Measures Mass of Single Molecule ScienceDaily (Aug. 26, 2012) http://www.sciencedaily.com/releases/2012/08/120826143528.htm

 

From 2009 a F1000 review http://f1000.com/prime/1162919 of: Towards single-molecule nanomechanical mass spectrometry. Naik et al.  http://www.nature.com/nnano/journal/v4/n7/abs/nnano.2009.152.html

 

Also by Roukes group in 2012 : Single-protein nanomechanical mass spectrometry in real time www.ncbi.nlm.nih.gov/pubmed/22922541

 

And associated comment in Nature http://www.nature.com/news/scaled-down-new-nano-device-can-weigh-single-molecules-1.11325

 

But if you don’t have access to Nature then this paper is freely available and taught me what a yoctogram is (10 ^-24 g)! 1.7 yg is the mass of a proton. Gosh.

A nanomechanical mass sensor with yoctogram resolution. Chaste et al. 2012 http://poggiolab.unibas.ch/full/2012MassSensor_Bachtold.pdf

 

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Identifying the margin:a new method to distinguish between cancerous and noncancerous tissue during surgery,

Identifying the margin: a new method to distinguish between cancerous and noncancerous tissue during surgery, 

 

Zoltan Takats, Julia Denes & James Kinross

Future Oncology
February 2012, Vol. 8, No. 2, Pages 113-116 , DOI 10.2217/fon.11.151

 

 

So if I understand this right, the concept is to use mass spec real time to guide surgeons between cancerous and healthy tissues. Wow, I am impressed! I am sure there is a LOT more work needed on databases of representitative tissues, data analysis and sampling still to be done. But I for one will be watching this closely, I think this has such potential. Neat work!

 

Key paragraph (a direct quote):

"Rapid evaporative ionization mass spectrometry was exclusively developed for intraoperative mass spectrometric investigation of tissues in vivo [19–21]. The technique is based on the discovery that surgical dissection techniques, such as electrosurgery or laser surgery, also act as an ionization method, such as converting molecular components of vital biological tissues into gaseous ions amenable to direct mass spectrometric analysis. The resulting data – featuring mainly complex lipid-type species – is highly similar to that provided by imaging mass spectrometry; hence, it is also tissue-specific. Combination of surgical tools with online mass spectrometric analyses resulted in the concept of so-called ‘intelligent knife’; a fully functional surgical cutting tool, which also analyses the dissected tissue parts. Tissue identification by the means of the rapid evaporative ionization mass spectrometry technique is based on comparison with authentic spectra, using multivariate statistical pattern recognition methods, including principal component analysis and linear discriminant analysis. As a result, online mass spectrometric information can be translated to histology-level identification of tissues on the timescale of a single second. Since then, while the intelligent knife concept was originally developed for electrosurgery [19], alternative surgical techniques (e.g., laser surgery and ultrasonic dissection [21,22]) were also successfully coupled with mass spectrometric analysis. The intelligent knife has the potential to revolutionize the identification of tumor margins in two fundamental ways: first, it can deploy an ‘alerting mode’ for the surgeon during the tumor resection and when working close to the solid tumor tissue. Whenever the tumor tissue is approached, the device gives alerts to the surgeon to lead the resection line further from the bulk tumor tissue. Infiltrations and proximal metastases induce positive tissue identification results, warning the surgeon of the presence of tumor cells in the resection line. This approach could potentially guarantee clear margins and, in principle, would result in minimized removal of healthy tissue. Mass spectrometric chemical profiling also allows the detection of tumor environment without actually cutting into the bulk tumor tissue, hence the risk of metastasis formation is not increased significantly by this approach. Second, the intelligent knife may be used in the so-called ‘microprobe mode’, where a miniaturized probe (not necessarily a surgical tool) is used for the evaporation (or other disintegrative sampling) of minute amounts of tissue. rapid evaporative ionization mass spectrometry technology enables the identification of as little as 50 µg of tissue material, so the sampling is minimally invasive. In this case, the surgeon, endoscopist, radiologist or therapeutic technician can sample any suspicious tissue feature on the surgical area and get histology-level identification within a single second."

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Tearing the Top Off ‘Top-Down’ Proteomics

Tearing the Top Off ‘Top-Down’ Proteomics | Mass Spectrometry Geekery | Scoop.it

A very nice article by Jeffrey Perkel in Biotechniques; it's a friendly easy going introduction to top down proteomics. Top down proteomics consists of measuring the intact mass of proteins to cover modifications and isoforms - often combined with fragmentation in the MS. Hold on for the description of impressive work by Jennifer Brodbelt (http://brodbelt.cm.utexas.edu/research/) who is developing photodissociation in Orbitraps.

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A home for raw proteomics data

A home for raw proteomics data | Mass Spectrometry Geekery | Scoop.it

The editorial in Nature Methods (9,419(2012)doi:10.1038/nmeth.201) discusses the problems that have hindered the deposition of raw proteomic data. These very problems – the accessibility of Tranche – have limited my own use of data repositories. So I am very happy to hear that EBI (http://www.ebi.ac.uk/) is moving to accept raw mass spec data files. It is timely for me personally: the image above is of my (shamefully) old fashioned solution to long-term storage of raw data files. As I type, these are being transferred to TGAC (http://www.tgac.ac.uk/) and I feel relieved. Next job is to pull out at least the published data sets, then I was planning to use Tranche but I will look west to EBI instead.

 

A final comment on the question ‘do people really want raw data files?’ Certainly I have received a few requests for my published data. Not frequently, but some. Similarly I have used and downloaded data from PRIDE or authors directly and occasionally been frustrated when it was not available.

 

I should  mention that I was very sad and sympathetic watching Tranche's fate: I really liked and admired its aims.

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Pain and joy of quantifying phosphorylation sites

Pain and joy of quantifying phosphorylation sites | Mass Spectrometry Geekery | Scoop.it

The mission was to relatively quantify phosphorylation sites on a protein after in vitro treatment with a panel of kinases. I had lots of spectra from previous work on the Orbitrap which had identified about 18 sites. (Some sites were considered to be ambiguous; I was sure that some peptides as a whole were phosphorylated, but unsure of the exact position as any of some neighbouring residues could be phosphorylated.) Luckily for me, an extremely talented post-doc had made lots of protein and done an excellent experiment: I had tones of material to work with.

 

The methods: Of course I used Skyline (MacCoss labs) to generate MRM methods of the Xevo from the Orbitrap data. I also wanted to test out positive/negative switching to see if I could identify new sites by the loss of -79 from phosphorylated peptides in nESI. The Xevo comes with a fancy predesigned template for this type of method; it combines polarity switching, with a quick precursor scan, then some MRM, then a product ion scan, before cycling back. Exciting stuff! I could not wait to get started.

 

The Pain: The pre-designed ‘phosphopeptide’ method appears to consistently* crash the mass spec such that it requires a full electronics re-start. Of course this happened at about 6 pm on a Friday night and to do so one needs to go round to the back of the instrument and put the vacuum pumps into ‘override’ so that you don’t vent the mass spec and add two more hours to your evening’s work. Then the electronics may be power cycled and the system restarted. OK, this is not a hard thing to do, but it is daunting with a new instrument and really not helped by small, hard to access switches (in our lab I have to wedge myself between two benches, try not to tread on the roughing pumps, nor crush or pull out any gas lines or cables and not knock over a gas cylinder). These elusive little sliver switches are shown in the picture above and the critical information – which leaver is the override and which turns off the electronics? – that information is finely engraved into the sliver panel by the green lights. Nice design job. Sure this is not something a user should do every day. But still occasionally we have to and believe me the adrenaline pumped as I made my choice. Getting is wrong would not be a flat-out disaster, but is something one should try to avoid.

*To be fair I only did this twice, but with two full system crashes on a Friday night with a recovery time of at least an hour, I called it a night. I have shared all this data with Waters, who advise ‘make the method simpler’. Sure enough, methods using ‘survey scans’ triggered from either -79 or the neutral loss of 98 do work.

 

The Joy: Once I abandoned the new fancy method and returned to MRMs the work went well. Out of my 18+ candidate sites, 10 could be reliably observed by MRM. I also monitored many unmodified ‘proteotypic’ peptides, and other peptides with and without oxidation of methionine and with various miscleavages. Why bother with these last two things? Well, phosphorylation sites are very inconsiderate and will often occur with additional modifications (oxMet being a common one) and their peptides frequently have ragged ends (sites where trypsin has two close choices RK and the like) or phosphorylation itself can influence how a peptide is cleaved. Many quantitative proteomic experiments avoid such modified or miscleaved peptides, but there the objective is generally to quantify the protein and use the peptides as surrogates. Here I was reasonably sure I had similar amounts of protein and was after relative change in phosphorylation status.

 

Well it worked, three replicated injections of each sample across the panel of 8 kinases and 10 phosphorylation sites showed that yes – even with a generous in vitro incubation of kinases and substrates - some sites are specifically phosphorylated.

This work was tremendously exciting for me, not just getting nice data from the new instrument, but seeing theory (that certain proteins may act as ‘hubs’ to integrate signals from many different pathways) come alive in my hands. Well that’s the sort of feeling that makes science, and late Friday nights, worthwhile. Of course we need to do more biological replicates (two down) and pull together a few more things, but the essence is there and the ‘oh gosh it actually worked!’ is enjoyable. Next challenge will be to move from in vitro to vivo…

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What's in the box? An ion trap!

What's in the box? An ion trap! | Mass Spectrometry Geekery | Scoop.it

The LTQ ion trap on our Orbitrap was replaced last week. Just this inner component, the essence of the linear ion trap and it has done us well since 2003 when we at TSL, first purchased the LTQ. A few years later, in 2007 we upgraded it to an orbitrap in collaboration with JIC and IFR. The LTQ part was taken away and fully overhauled but I think this little ion trap was exactly the same one.

 

So this in what those inner quadrupoles look like and the gloved hands give a nice sense of scale. I would have liked to keep this for teaching purposes, but sadly the old component had to go back and instead we have a little set of photographs (thanks to Jan). I think the LTQ is a great robust design, it has simply worked (well, nearly all of the time, as much as one can reasonably expect from a complex instrument in 24/7 operation).

 

I would have liked to link to a nice little teaching video but can only offer you the obvious wiki page (http://en.wikipedia.org/wiki/Mass_spectrometry) or a highly detailed paper (http://www.chem.mun.ca/courseinfo/c4151/references/Linear%20ion%20trap.pdf). This wiki gap should be plugged really - ion traps are so well established and such a fundamental part of research and analytical sciences.

 

So instead I will enthuse briefly and (I hope) at a reasonable level of detail:

Looking at the trap reminds me both of the achievements of mass spec; that signals as low as in the order of hundreds of thousands of ions (charged molecules) may be detected. That is pretty amazing, 500,000 is a number that I can quite clearly imagine - and much smaller than current economic crisis figures for example (http://xkcd.com/980/) - and only a tiny fraction of a mole (chemical unit 6x10^23). Yet with all the current excitement about single molecule detection in microscopy and DNA sequencing I enviously think of what might be a hard limit for mass spec. You see, a mass spec must actually move ions physically in order to detect them, over really quite considerable distances, and in doing so loses some en route. Furthermore, when looking at the fragmentation of a molecule you need to have a population of ions: if it breaks into two pieces each piece must retain at least one charge to be detectable – uncharged molecules are invisible to a mass spec. So to fully sequence a peptide of eight amino acids one needs at least seven specific fragments (as one knows the total mass), preferably more – observing matching parts of a fragmented molecule (b and y ions to use some jargon) is much more convincing than observing just a few pieces. A spectra is rarely completely free of noise, so many fragments need to be recorded to give some reasonable intensity. So I find it hard to imagine, even with perfect collection and trapping of ions, that this limit of needing a population of ions to fragment can even be over come by mass spec.

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Patently Limited

The arguments for and against patents have raged long and fierce and more knowledgeable people that I argue eloquently for each case. In general I am in favour of rewarding invention and am a somewhat uneasy ‘inventor’ on a genetic patent myself. However, the longer I watch the mass spectrometry market, the more frequently I think that the development of instruments is limited by patent infringement. I visualise a kind of developmental landscape where the optimal peak – or instrument configuration – is unobtainable or delayed because of our legal and financial systems. So I came across this snippet, with a dreadful title, from ProteoMonitor "In PerkinElmer Mass Spec Suit, Waters' Patent Reexam Requests Could Signal Infringement Worries" with a feeling of fatigue. [Apologies that the full text is only available to subscribers (free) I couldn’t quickly find an equivalent story.]

 

In essence the development under dispute allows more ions to be captured from the spray resulting in an increase of sensitivity (or at least that is how I understand it). The title of these patents is wonderfully vague “Multipole ion guide for mass spectrometry” but the applications (I think) have been used for the ‘step wave’ as Waters calls the latest developments on the Xevo and Synapt. And conceptually similar developments have been seen recently in ThermoScientific instruments as ‘ion funnels’. Full details of the Perkin Elmer patents are here (http://www.patentstorm.us/patents/5652427.html, http://www.patentstorm.us/patents/5962851.html). I had been welcoming the proliferation of ion guides, funnels, call-them-what-you-will, as a release from such a limitation.

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Kalibr's comment, April 11, 2012 10:52 AM
And on the same topic, Perkin also sued Agilent.

Maybe the Micromass people that got into Waters didn't learn from the previous law suit (by Applied Biosystem)
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Waters: An Overview of the Principles of MSE

Waters: An Overview of the Principles of MSE | Mass Spectrometry Geekery | Scoop.it

This note (pdf) by Waters explains one of the main features of the Synapt pretty well (with only a little sales blurb in the introduction); the MSE technique. In a typical proteomic MS/MS experiment the instrument switches between looking at all precursor masses (ok over a defined mass range) and then selecting the top 5 or so for specific, sequential fragmentation scans. Waters take a different approach, abandoning the selection of a precursor ion for individual fragmentation and instead fragmenting everything.The clever bit is then to use the elution profile of precursor masses from the liquid chromatography component to deconvolute the mess of fragments from many precursor ions at once.

 

This technique has been around for a while now. I must confess that I have been pretty skeptical about it over the years, but I am coming around to it. There have been several good talks at conferences in the last year or  so and more and more labs are using it. My doubts were around how much one can really deconvolute if 50 or more precursor ions are fragmented all at once, they would give an extremely complex combined fragmentention spectrum. I talked about this at length with Waters during our purchase and yes, of course there are limits. In particular we hope that another new feature, the ion mobility cell, which adds another dimension to the separation, will add more depth. I admit that I am probably biased in my thinking by my long experience with ion traps. So I enjoy challenging my doubts.

 

Here at NRP, Gerhard and Jan have been analysing reasonably complex mixtures, co-immunoprecipitated proteins from 1D gel slices,  on both the Orbitrap and the Synapt. From early datasets it looks like the performance of both instruments for the top proteins is similar. I am delighted to discover that one of my favourite analytical programs, Scaffold (Proteome Software), can represent the co-eluting precursor ions as blobs over time - as you can see in the picture above - the pink large ones are identified peptide ions, the small blue ones unidentified co-eluting ions. Scaffold also provides nicely annotated, deconvoluted spectra for teh identified peptides. How to present MSE fragmentation data to reviewers had been another of my worries. (As reviewers, like myself, might be expecting neat selected precursor ion fragmentation spectra).

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Kalibr's comment, April 11, 2012 10:48 AM
Hi Alex!

Just found your site. Congrats. I'm reading through. On this topic I would suggest reading the following Abersold paper:

http://www.mcponline.org/content/early/2012/01/18/mcp.O111.016717.long
alex's comment, April 12, 2012 5:27 AM
Hi Kalibr,

Welcome and thank you for commenting :)

Yes good reference, SWATH MS cleverly adapts the old idea of sectioning up MS/MS acquisition by mass range and then realises the full potential by applying the knowledge from known proteomes. Neat stuff. I was lucky enuogh to see Natalie Selevek talk about this at a conference (PMMX) just a couple of weeks ago.
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A mass spectrometry–guided genome mining approach for natural product peptidogenomics : Nature Chemical Biology : Nature Publishing Group

A mass spectrometry–guided genome mining approach for natural product peptidogenomics : Nature Chemical Biology : Nature Publishing Group | Mass Spectrometry Geekery | Scoop.it

An inspriational paper, using both MALDI imaging of bacterial colonies (growing directly on the target plate) and de novo sequencing tagging to almost casually identify dozens of novel natural products.

 

 

Abstract

Peptide natural products show broad biological properties and are commonly produced by orthogonal ribosomal and nonribosomal pathways in prokaryotes and eukaryotes. To harvest this large and diverse resource of bioactive molecules, we introduce here natural product peptidogenomics (NPP), a new MS–guided genome-mining method that connects the chemotypes of peptide natural products to their biosynthetic gene clusters by iteratively matching de novo tandem MS (MSn) structures to genomics-based structures following biosynthetic logic. In this study, we show that NPP enabled the rapid characterization of over ten chemically diverse ribosomal and nonribosomal peptide natural products of previously unidentified composition from Streptomycete bacteria as a proof of concept to begin automating the genome-mining process. We show the identification of lantipeptides, lasso peptides, linardins, formylated peptides and lipopeptides, many of which are from well-characterized model Streptomycetes, highlighting the power of NPP in the discovery of new peptide natural products from even intensely studied organisms.

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Rapid empirical discovery of optimal peptides for targeted proteomics : Nature Methods : Nature Publishing Group

Rapid empirical discovery of optimal peptides for targeted proteomics : Nature Methods : Nature Publishing Group | Mass Spectrometry Geekery | Scoop.it

"An empirical approach for identifying optimal proteotypic peptides and fragmentation patterns from in vitro-synthesized proteins, for targeted proteomics applications, is described."

 

Using cDNA libraries to generate peptides to develop MRM transitions sounds like a great idea for those targets without previous spectral data.

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