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Rationally designed synthetic peptide as versatile calibrant to improve the accuracy of protein sequence analysis using MALDI mass spectrometry

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Chinese Chemical Letters 2024年第3期35卷 214-220页

作者： Lingpeng Zhan Yanyi Huang Guanbo Wang Institute for Cell Analysis Shenzhen Bay LaboratoryShenzhen 518132China Biomedical Pioneering Innovation Center Peking UniversityBeijing 100871China

Matrix-assisted laser desorption/ionization(MALDI)mass spectrometry(MS)plays an indispensable role in analyzing protein covalent structures.The reliable identification of amino acid residues and modifications relies on the mass accuracy,which is highly dependent on calibration.However,the accuracy provided by the currently available calibrants still needs further improvement in terms of compatibility with multiple tandem MS modes or ion polarity modes,calibratable range,and minimizing suppression of and interference with analyte signals.Here aiming at developing a versatile calibrant to solve these problem,we designed a synthetic peptide format of calibrant R_x(GDP_n)_m(referred to as“Gly-Asp-Pro,GDP”)according to the chemical natures of amino acids and polypeptide fragmentation rules in tandem MS.With four types of amino acid residues selected and arranged through rational designs,a GDP peptide produces highly regulated fragments that give rise to evenly spaced signals in each tandem MS mode and is compatible with both positive and negative ion modes.In internal calibration,its regulated fragmentation pattern minimizes interference with analyte signals,and using a single peptide as the input minimizes suppression of the analyte signals.As demonstrated by analyses of proteins including monoclonal antibody and Aβ-42,these features allowed significant increase of the mass accuracy and precision,which improved sequence coverage and sequence resolution in sequence analyses(including de novo sequencing).This rational design strategy may also inspire further development of synthetic calibrants that benefit structural analysis of biomolecules.

关键词： Biomolecule design Synthetic peptide protein sequencing Covalent structure De novo sequencing Mass spectrometry Gas-phase fragmentation

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Adaptive nanopores:A bioinspired label-free approach for protein sequencing and identification

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Nano Research 2021年第1期14卷 328-333页

作者： Andrea Spitaleri Denis Garoli Moritz Schutte Hans Lehrach Walter Rocchia Francesco De Angelis CONCEPT Lab Istituto Italiano di TecnologiaVia Morego 30Genova1-16163Italy Center for Omics Sciences IRCCS San Raffaele Scientific InstituteMilanVia Olgettina 58Milano1-20132Italy Plasmon Nanotechnology Unit Istituto Italiano di TecnologiaVia Morego 30Genova1-16163Italy AB ANALITICA s.r.l Via Svizzera 161-35127 PadovaItaly Alacris Theranostics GmbH Max-Planck-Strasse 3D-12489 BerlinGermany Max Planck Institute for Molecular Genetics Ihnestrasse 63-73D-14195 BerlinGermany

Single molecule protein sequencing would tremendously impact in proteomics and human biology and it would promote the development of novel diagnostic and therapeutic approaches.However,its technological realization can only be envisioned,and huge challenges need to be overcome.Major difficulties are inherent to the structure of proteins,which are composed by several different amino-acids.Despite long standing efforts,only few complex techniques,such as Edman degradation,liquid chromatography and mass spectroscopy,make protein sequencing possible.Unfortunately,these techniques present significant limitations in terms of amount of sample required and dynamic range of measurement.It is known that proteins can distinguish closely similar molecules.Moreover,several proteins can work as biological nanopores in order to perform single molecule detection and sequencing.Unfortunately,while DNA sequencing by means of nanopores is demonstrated,very few examples of nanopores able to perform reliable protein-sequencing have been reported sofar.Here,we investigate,by means of molecular dynamics simulations,how a re-engineered protein,acting as biological nanopore,can be used to recognize the sequence of a translocating peptide by sensing the MshapeH of individual amino-acids.In our simulations we demonstrate that it is possible to discriminate with high fidelity,9 different amino-acids in a short peptide translocating through the engineered construct.The method,here shown for fluorescence-based sequencing,does not require any labelling of the peptidic analyte.These results can pave the way for a new and highly sensitive method of sequencing.

关键词： nanopores single molecule sequencing protein sequencing luorescence resonance energy transfer(FRET) amino-acids fluorescence

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