title: An improved stable isotope N-terminal labeling approach with light/heavy TMPP to automate proteogenomics data validation: dN-TOP.
abstract: In silico gene prediction has proven to be prone to errors, especially regarding precise localization of start codons that spread in subsequent biological studies. Therefore, the high throughput characterization of protein N-termini is becoming an emerging challenge in the proteomics and especially proteogenomics fields. The trimethoxyphenyl phosphonium (TMPP) labeling approach (N-TOP) is an efficient N-terminomic approach that allows the characterization of both N-terminal and internal peptides in a single experiment. Due to its permanent positive charge, TMPP labeling strongly affects MS/MS fragmentation resulting in unadapted scoring of TMPP-derivatized peptide spectra by classical search engines. This behavior has led to difficulties in validating TMPP-derivatized peptide identifications with usual score filtering and thus to low/underestimated numbers of identified N-termini. We present herein a new strategy (dN-TOP) that overwhelmed the previous limitation allowing a confident and automated N-terminal peptide validation thanks to a combined labeling with light and heavy TMPP reagents. We show how this double labeling allows increasing the number of validated N-terminal peptides. This strategy represents a considerable improvement to the well-established N-TOP method with an enhanced and accelerated data processing making it now fully compatible with high-throughput proteogenomics studies.
authors: Laboratoire de Spectrométrie de Masse BioOrganique, IPHC, Université de Strasbourg, CNRS, UMR7178, Strasbourg, France.
authors: Diego
authors: D
authors: Bertaccini
authors: Sebastian
authors: S
authors: Vaca
authors: Christine
authors: C
authors: Carapito
authors: Florence
authors: F
authors: Arsène-Ploetze
authors: Alain
authors: A
authors: Van Dorsselaer
authors: Christine
authors: C
authors: Schaeffer-Reiss
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000032:analysis* / Q000737:chemistry; D002853:Chromatography, Liquid / Q000592:standards*; D007553:Isotope Labeling / Q000379:methods*; D008969:Molecular Sequence Data; D009943:Organophosphorus Compounds / Q000737:chemistry; D010446:Peptide Fragments / Q000302:isolation & purification*; D017434:Protein Structure, Tertiary; D020560:Proteobacteria / Q000737:chemistry / Q000254:growth & development; D040901:Proteomics; D055672:Static Electricity; D053719:Tandem Mass Spectrometry / Q000592:standards*
chemical_list: D001426:Bacterial Proteins; D009943:Organophosphorus Compounds; D010446:Peptide Fragments