Thursday, October 22, 2015

MultiNotch MS3 Quantitation Solution Developed for Proteome Sciences

The PEAKS development team has developed a solution to address MultiNotch MS3 Quantitation, currently in use by Proteome Sciences for biomarker discovery. MS3 provides more accurate quantification for isobaric tagging experiments, specifically improving TMT and iTRAQ based studies.

In 2011, Gygi et al. reported isobaric labeling mass spectrometry–based proteome quantification commonly suffers ratio distortion owing to protein quantification interference.1 A more recent publication by the authors identifies that the combination of quantitative isobaric reagents and the MultiNotch MS3 method improves the dynamic range of reporter ion quantitation, reducing reporter ion signal variance, and ultimately produces more high-quality quantitative measurement.2 Our development team implemented a data analysis solution to address these points in order to generate more sensitive and accurate results for end users.

The MultiNotch MS3 quantitation solution is currently in use by Proteome Sciences.

Wednesday, August 19, 2015

New Methods of Peptide Footprinting

Earlier this year Methods published an article entitled Using Hydroxyl Radical Footprinting to Explore the Free Energy Landscape of Protein Folding. The article provides guidelines to ensure best practises when protein mapping using the emerging technology called fast photochemical oxidation of proteins (FPOP). Recently, BSI spoke with lead author, Dr. Antonio Calabrese from the University of Leeds to discover more about his research.

Understanding structure, flexibility and dynamics of side chains in different protein conformers is critical to structural biologists. While very successful for other applications, nuclear magnetic resonance (NMR) is limited in its ability to perform direct comparison on partially folded proteins due to conformational exchange; thus other methods are required.
Dr. Antonio Calabrese, University of Leeds
Dr. Antonio Calabrese,
University of Leeds

FPOP is a novel mass spectrometry based method for protein footprinting that has the ability to characterize protein variants in different conformational states. This is achieved by determining conformational changes in proteins caused by folding/ unfolding, with the added capability of investigating changes in solvent accessibility that accompany ligand binding. Further, the speed of FPOP avoids conformational averaging. According to Dr. Calabrese:

The lab is very interested in method development, developing new techniques and applying a variety of mass spectrometry technique to study protein structure. We also work with native mass spectrometry and chemical cross-linking and so… labeling with hydroxyl radicals is complementary to hydrogen deuterium exchange (HDX). One of the things different about it is because you have a covalent label you can be a bit more rigorous with your downstream sample handlings.

Within the realm of protein mapping, a significant interest of the group is surface mapping. To accomplish this, the surface area of a protein is labelled with hydroxyl radicals; opening the opportunity to identify labelling sites at the amino acid level and perform quantification on the observed oxidation.

Dr. Calabrese was not shy about addressing the challenges of FPOP:
The thing that is rather complex about the data that you get out of FPOP is that generally speaking you are looking at modifications which are quite lowly abundant and so being able to reliably quantify those levels of oxidation and quantify those modifications is often quite challenging; especially if you want to analyze your data in order to obtain very specific information…. We used PEAKS to help assign our modification sites and then we also had to manually validate, specifically the modification, sites.
Peptide Quantification
Peptide level quantification of oxidation levels.

In addition to using PEAKS for identification, the quantification node, PEAKS Q, was applied in both label and label free techniques. Precursor Ion Quantification (traditionally used for ICAT/SILAC) was configured for custom variable modification mining and Label Free Quantification for the remainder of features. When asked about satisfaction from the automated results produced by PEAKS the answer was very positive:

The automated results were definitely quite good. Whenever we manually validated those automated results they were pretty much spot on. When results are right it is obviously much easier to work with. Also, the capacity to look at the de novo spectra means you have got a lot more data to play with; otherwise much of it just gets thrown away. […] Now we are using it routinely for all of our DDA data analysis in the lab.

As with many other emerging technologies, the project is set to progress onto other complicated systems where it can continue to be complementary to HDX.

We have just looked at some relatively simple systems [soluble proteins], but now we are starting to look at more complicated systems using the techniques; so now are starting to look at characterizing membrane proteins which are obviously a lot more challenging and trying to optimize everything now.

We are grateful to have had the opportunity to speak with Dr. Antonio Calabrese and receive insight into emerging methods that dig deeper into explaining our proteomic world.

For those interested in learning more about the PEAKS software, regularly scheduled public webinars are available for free with our support team here.

Friday, July 3, 2015

PEAKS Training Workshop: Stanford, California

The PEAKS Training Workshop is coming to Stanford University (Stanford, California) on August 24-25.

The Training Workshop provides a full hands-on experience with PEAKS 7.5 for both new and experienced users of PEAKS Studio. In addition to demonstrating the logic and algorithm behind PEAKS software, the workshop focuses on how PEAKS may be used specifically toward the users’ research and applications.

Participants of the course can expect to increase their knowledge of LC-MS/MS data analysis and result validation upon completion of the course. Visit our training website for more details and to sign up.

Tuesday, June 16, 2015

PEAKS Studio 7.5

We are excited to announce the release of PEAKS Studio 7.5. There are many new features in this release that expand the capabilities for our users. Just to name a few favourites, consider:

  • PEAKS PTM Profiling – to detect and quantify modifications
  • Peptide Mapping - a powerful comparison tool, providing an intensity map of all the peptides associated with any particular protein.
  • Peptide Feature Intensity – in one click users can analyze and quantitative peptides across all samples.
  • Automated Multi-Round Search – automatically filter out high quality spectra unmatched after database searching perform additional searches against different sequence databases or change parameter settings to help explain more from your data.
These were designed to further compliment PEAKS’ proven track record of high accuracy and high sensitivity in third party studies. A complete list of improvements and additions to the software can be found here: PEAKS Studio 7.5.

Monday, June 15, 2015

ASMS 2015

The expanding popularity of PEAKS made for another exciting ASMS annual conference. During the PEAKS Meeting on Sunday, BSI gave an early glimpse into a future version of PEAKS Studio. Guest speakers also presented current research projects where PEAKS proved vital:

  • Jeanne Rumsey of Washington University in St. Louis,
  • Database Search Strategies for Biomarker Design in Infectious Diseases
  • Dr. Cheryl Lichti, University of Texas Medical Branch,
  • PEAKS and the Quest for Single Amino Acid Variants

During the conference a number of notable oral presentations and posters resonated well with attendees and emerging methods. (Please note you will need to login using your ASMS details to view the presentations.) A significant list of oral presentations and posters relevant to PEAKS users are available on our ASMS 2015 webpage. Our own poster, Peptide Mapping for Quantitative Profiling of Peptide Variants using PEAKS is also available.

Wednesday, April 15, 2015

ABRF iPRG 2015

The purpose of this year’s ABRF iPRG study was to evaluate different data analysis methods for relative protein-level quantification in label-free proteomics. In conclusion of the study, PEAKS label-free quantification proved to successfully demonstrate a high-level of accuracy in determining relative protein abundance.
Graphs from the results were retrieved from the ABRF iPRG Preliminary Results Summary. To view the full Preliminary Results Summary, please use the link provided below.
The first graph shown is an overview of all the participating Study IDs in relation to their results as seen on slide 20 in the Preliminary Results Summary. The same graph is then presented again on slides 24 and 25 (zoomed in), to show the specific tools used for peptide ID, as well as quantification for each submission. .
Of the 51 submissions, very few were able to recognize the true differentially abundant (DA) proteins, as seen in the graph. PEAKS, however, recognized all 6 true DA proteins (represented in blue), and reported 0 false DA proteins (represented in red).
Participating researchers were required to detect and identify proteins that changed in relative abundance among four biological samples, prepared from the same S. cerevisae yeast cell lysate.
To truly focus on the statistical assessment of the varying data processing approaches, the peptide identifications and their associated integrated peak intensities were provided to all participants.

Wednesday, March 11, 2015

PEAKS in Germany: Proteomic Forum 2015

Come by and visit PEAKS at this year's DGPF: Proteomic Forum (booth 19). The conference will be taking place March 22-25 at the Technical University Berlin (Germany).

In addition, PEAKS will be hosting a Training Workshop, March 19-20, 2015, at the Crowne Plaza, Berlin City Center. The workshop is designed to give users a hands-on approach to fully understanding the capability of PEAKS. Visit for more information and registration.

“The German Society of Proteomic Research coordinates and provides a network comprising academic institutions, commercial companies and special institutions at the national and international level in order to support the extension of scientific knowledge in the field of proteome research and protein analysis.”

For more information, visit