Advancing knowledge requires taking risks. However, novel biology is often a prerequisite for publication, and this requirement is currently holding scientists back from taking risks. This is stalling scientific progress. Yet, every experiment that is built on a solid experimental design and explored with robust data analysis techniques is equally valuable to the advancement of knowledge. This is why EuPA Open Proteomics provides an open platform to share good experiments within the field of proteomics. Here, novel biology is a by-product of good experimentation. EuPA Open Proteomics welcomes:
Team: Peptidal Investigators Method: Sample preparationDilution of peptide mixture in 0.1% TFA and preparation of aliquots corresponding to approx.80 fmol per peptide LC-MS/MS analysis Ultimate 3000 nano RSLC system coupled to an Orbitrap Fusion Lumos mass spectrometer (both Thermo scientific) 100 μm × 2 cm pre-column/75 μm × 50 cm C18 analytical column (PepMap RSLC, Thermo Scientific) 45 min gradient 3-50% solvent B (solvent A: 0.1% FA, solvent B: 84% Acetonitrile, 0.1% FA)Top speed acquisition (3 s) followed by creation of an inclusion list for subsequent runs
Using primarily LC-MS/MS de novo sequencing techniques, we concluded that the peptides form the following sentence from Sir JJ Thomson's preface to Rays of Positive Electricity and Their Application to Chemical Analyses: "I feel sure that there are many problems in chemistry which could be solved with far greater ease by this than by any other method. The method is surprisingly sensitive, more so even than that of spectrum analysis, requires an infinitesimal amount of material, and does not require this to be specially purified." Here, we detail our process for deciphering the peptide mixture composition and finding the sentence they form and from what book the sentence comes from.
In the recent year, we felt like we were not truly showing our full potential in our PhD projects, and so we were very happy and excited when YPIC announced the ultimate proteomics challenge. This gave us the opportunity of showing off and procrastinating at the same time:) The challenge was to identify the amino acid sequence of 19 synthetic peptides made up from an English text and then find the book that it came from. For this task we chose to run on an Orbitrap Fusion™ Lumos™ Tribrid™ Mass Spectrometer with two different sensitive MS2 resolutions, each with both HCD and CID fragmentation consecutively. This strategy was chosen because we speculated that multiple MS2 scans at high quality would be beneficial over lower resolution, speed and quantity in the relatively sparse sample. The resulting chromatogram did not reveal 19 sharp distinct peaks and it was not clear to us where to start a manual spectra interpretation. We instead used the de novo option in the MaxQuant software and the resulting output gave us two phrases with words that were specific enough to be searched in the magic Google search engine. Google gave us the name of a very famous physicist, namely Sir Joseph John Thomson, and a reference to his book “Rays of positive electricity” from 1913. We then converted the paragraph we believed to be the right one into a FASTA format and used it with MaxQuant to do a database search. This resulted in 16 perfectly FASTA search-identified peptide sequences, one with a missing PTM and one found as a truncated version. The remaining one was identified within the MaxQuant de novo sequencing results. We thus show in this study that our workflow combining de novo spectra analysis algorithms with an online search engine is ideally suited for all applications where users want to decipher peptide-encoded prefaces of 20th century science books.
