PTMScan Direct

The front page of MCP is back online.  One reason that this is good is that you can read about PTMScan Direct.  This paper is from Cell Signaling and describes a really good combination of using antibody enrichment and mass spectrometry.
The PTMScan technology is based on mixtures of antibodies that can be used to pull down multiple important signaling pathway proteins all at once.  The pulldowns are then analyzed by LC-MS/MS (in the paper, they describe the use of an LTQ-Orbitrap Velos).  The antibody combinations can provide you with a rapid and thorough look into what pathways are involved in your system of interest.  The technology is fully compatible with both label free discovery methods, as well as SILAC for quantification.
My one small criticism with this absolutely brilliant paper is in the settings they used in their MS/MS analysis.  I was very surprised to see that they used an MS1 mass window of 50ppm with an MS/MS window of 1 Da.  These are considerably less strict than what I use and what I am used to seeing in the literature.  When our bionformatician informed me that our false discovery rate calculations kirunefited from using larger mass windows, I began using 10ppm and 0.5 Da with our Velos.  I don't think I could be convinced to widen my MS1 window 5-fold.  My only thought on why they used these settings is that the Sorceror system they used for analysis kirunefits dramatically from having a larger window, but at this point I'm just speculating.

In summary, this technique takes immunoaffinity pull-downs to a whole new level.  I expect this technology to be in high-demand, particularly in drug mechanism studies.  If you read one paper this month, this is the one I recommend.

READ MORE - PTMScan Direct

Metabolic Fate of Tea Polyphenols in Humans

This new paper in JPR isn't really a proteomics paper, its more of a metabolomics (metabonomics) study, but it is definitely interesting enough to warrant a quick mention.
In the study, 20 healthy men and 20 healthy women were put on a specific polyphenol-free diet (no caffeine or chocolate for 6 weeks).  The exception to this diet was the daily administration of a concentrated tea that was the equivalent of ~5 cups of commercially available tea.  The participants then submitted urine samples over a rigorous schedule, with some participants submitting samples 6 times daily.
The urine samples were analyzed with LC-MS using a Waters ACQUITY UPLC system coupled to a Micromass Q-TOF.  The samples were also analyzed by GC-MS using an Agilent 6890N GC and a Pegasus HT TOF system.
The study determined the rate of clearance of the polyphenols present in these teas as well as the appearance of compounds that resulted from metabolism of these molecules.  One interesting fact the study found was that although caffeine appearance in the urine peaked 1 hour after the tea was ingested, it didn't completely clear the body until 9 hours post ingestion.
The most interesting part of this study, in my opinion, was the use of multivariate statistics for results analysis.  I am encouraged when I see the science of statistics infiltrating the analysis of MS data.  Its interesting to me how rare this has been so far, especially considering how essential statistics is to genomics.

READ MORE - Metabolic Fate of Tea Polyphenols in Humans

Skyline MS1 Filtering and MS1 Quantitation

This paper currently in press at MCP is from a group of researchers at the Buck Institute for Research on Aging.  The paper details a new cross-platform software package for quantitative proteomics that they call Skyline.  (Not to be confused with the legendary Japanese car that was illegal to import into the U.S. due to the engineers focusing so completely on making the car fast that they forgot to include any concern for occupant safety)

(Not this Skyline)

No, this Skyline is an attempt to remove one set of variables from quality control in label free quantitative proteomics experiments.  The Skyline software aims to be a reference system for the analysis and meta-analysis of data generated on any MS platform.  In this text, they demonstrate the analysis of data from a QStar Elite, a TripleTOF 5600, and an LTQ FTICR.  This is accomplished without first converting the RAW data to one of the generic accepted formats.

In order to show the true power of Skyline, the authors investigate two data sets, one generated from the enrichment of lysine-acetylated peptides and a second from TiO2 enriched phosphopeptides.  By walking us through these analyses, the authors show us both the power of Skyline as well as a walkthrough of the software and its features.  

Now, this is a nice paper, and I am looking forward to re-running some label free quant data I previously analyzed against Skyline, but this isn't the most interesting part of this paper for me.

The most interesting part is in the introduction to this paper when the authors describe their unpublished attempts to use iTRAQ to perform quantification of the acetylome.  They found that iTRAQ labeling did not occur (or at least not efficiently) on peptides that were acetylated on lysines.  For that reason, they stopped using iTRAQ and focused on label free quant of the acetylome.  I wonder if the efficiency of iTRAQ labeling could be used as an indirect way to measure global changes in the acetylome the same way we once used cys-TMT to measure the global alkylation of cysteines?

READ MORE - Skyline MS1 Filtering and MS1 Quantitation

MCP frontpage down

If you've tried to access the new issue of MCP through the Highlights page, I'm sure you noticed that all of the links are down.  I alerted Andrew Harmon at MCP of the problem this morning, and they are working to resolve it.  Hopefully they'll get it fixed soon, they have some great stuff in this issue!

READ MORE - MCP frontpage down