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Rescooped by Drew Gibson from Mass Spectrometry Geekery
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Designing transitions: Skyline

Designing transitions: Skyline | drew-gibson-mass-spec | 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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mMass - Open Source Mass Spectrometry Tool

mMass - Open Source Mass Spectrometry Tool | drew-gibson-mass-spec | 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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Waters: An Overview of the Principles of MSE

Waters: An Overview of the Principles of MSE | drew-gibson-mass-spec | 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.