组合质谱使基本的蛋白质鉴定成为可能：使用LTQ Orbitrap质谱对完整蛋白质进行质量分析和自上而下的碎裂

简介：

A fundamental stage in protein characterization is to determine and verify the intact state of the macromolecule. This is often accomplished through the use of mass spectrometry (MS) to first detect and measure the molecular mass. Beyond confirmation of intact mass, the next objective is the verification of its primary structure, the amino acid sequence of the protein. Traditionally, a map of the macromolecule is reconstructed from matching masses of peptide fragments produced through external enzymatic digestion of the protein to masses calculated from an in silico digest of the target protein sequence. A more direct approach involves top-down MS/MS of the intact protein molecular ion.

仪器：

Thermo Scientific LTQ Orbitrap XL or LTQ Orbitrap Velos hybrid mass spectrometer

总结：

•The high resolving power of Orbitrap analyzer detection llows isotopic resolution of proteins as large as 47 kDa as well as large fragments, facilitating top-down MS/MS xperiments.

• For proteins larger than 50 kDa (for which isotopic esolution is not possible) a resolution setting of 7500 or 15,000 should be used for Orbitrap analyzer detection o improve the signal-to-noise ratio of the mass spectra.

• High-mass-accuracy detection offered by the Orbitrap nalyzer allows more precise measurement of molecular masses of intact proteins.

• Proper desalting and desolvation are necessary for cleaner rotein signals and better signal-to-noise ratios. Proper setting for temperatures, desolvation gases and source oltages are necessary for the removal of unwanted on-covalent adducts characteristic of large proteins.

• A reduced detection delay should be used for best etection of large macromolecules in LTQ Orbitrap ass spectrometers.

• The availability of multiple activation strategies in conjunction ith high-performance detection characteristics offered by the hybrid systems allows top-down protein haracterization with more complete sequence definition. ETD, in particular, offers amino acid resolution for large arts of the N and C termini of the sequence.

• Reducing the gas flow from the HCD collision cell for LTQ Orbitrap XL instruments (turning the gas off) esults in improved detection of large proteins in the rbitrap analyzer.