Quantifying protein interaction dynamics by SWATH mass spectometry: application to the 14-3-3 system

A recent article in Nature Methods describes a very nice study on mapping protein-protein interactions using mass spectrometry.  The proteins of interest are the 14-3-3 family of generic scaffolding proteins.  The MS method used is a data independent acquisition (DIA) process where compiled spectral libraries are compared to affinity enriched samples.  The researchers do a fantastic job of showing how to set up an affinity enriched study with mass spectrometry.  The controls and statistics are all top-notch, and the study absolutely deserves to be in a journal this prestigious and you should totally check it out (it's open access here!)

In the paper, the authors defend their use of their DIA method rather strongly, noting the relative weaknesses of data dependent acquisition (DDA) approaches, like the TopN method, in terms of the number of fragmentation events that can be achieved per unit of time.  This is undoubtedly true.  A TopN method won't fragment all the ions in a run, but what it does end up fragmenting will suffer from considerably less co-isolation effects than any DIA experiment, and therefore will have a markedly higher identification efficiency and lower false discovery rates.  Also, the effects of peptide undersampling in DDA will be considerably less of a disadvantage in an affinity enrichment than in a global peptide sample.  Given the number of peptides/proteins evaluated in this study, there is no reason that it could not have been performed with a standard DDA approach.

Another reason that DIA was used here was the relative dynamic range of that approach.  The authors found that they could accurately quantify within 4 orders of magnitude.  This is an impressive number for a peptide study, but still easily within the 4.5 orders achievable with an Orbitrap.

Don't get me wrong.  This is an awesome paper!  From an analysis perspective this is as nice of a protein interaction study that I have ever seen.  I just don't want anyone to look at this and think that they can only reproduce this level of work using DIA.  The gold in this paper is the experimental setup and the statistical rigor employed.  From the instrumentation standpoint, this study could be reproduced with virtually any modern mass spectrometer, and if you're lucky enough to have an Orbitrap you could do this study with better dynamic range and simpler statistics than what had to be done here due to the relative inaccuracy of the instrument used.

TL/DR:  Great experimental setup in this paper in Nature Methods!  DIA may have been used here because it's pretty trendy right now.

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