Friday, November 24, 2017

Longitudinal quality assessment of iTRAQ labeled samples for clinical proteomics!

Have we underestimated the power of reporter ion based quantification (iTRAQ/TMT) in terms of long term clinical studies? This new study at JPR suggest we might need to reconsider it!

Over a 7 month period, this team collected xenograft samples. In all experiments they used a single reference sample and use that reference channel for normalization and in a lot of the other downstream work.

They demonstrate a surprising amount of power and reproducibility over this time. This is despite the use of 2D offline fractionation (via high pH reversed phase) into 24 fractions and keeping the fractions stored at -80C.  (96 fractions were collected, but they were concatenated into 24, using the same concatenation pattern for every fractionated sample -- for example, fractions 1,25,49 & 73 were combined for every set of peptides)

All the work for this study is performed on the same Orbitrap Velos instrument using a 2Da MS/MS isolation window on a top10 experiment with HCD fragmentation set at 35.

Interesting touch here is a confidence metric that takes into account the hydrophobicity of the peptides and the reporter ion intensity.

The real star of this huge amount of work (at least for me) is the 22 separate metrics that were tested to determine the most reproducible way to roll the reporter ion and PSM data up to the protein level. As hard as this Orbitrap was working, it looks like the statisticians may have put in more time.  In the end, they conclude that if you put the work in and do it right, there is no reason you can't utilize reporter ion quan in large scale clinical studies!

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