The science of measuring proteins
Intact protein analysis is performed for a number of reasons, including determination of molecular mass (during top-down analysis) and posttranslational modification (PTM) (e.g., monoclonal antibody glycosylation). Full characterization of intact proteins is often performed on proteins that have been enriched or purified. These proteins can be introduced into the mass spectrometer (MS) by either direct infusion or through liquid chromatography (LC) coupled with electrospray ionization (ESI).
The primary advantage of direct infusion is that it allots more time for signal averaging. However, most intact protein mixtures require separation prior to introduction into the MS to reduce precursor spectral complexity and minimize ion suppression. This separation can be performed by either on- or off-line LC. High-resolution accurate mass (HRAM) analysis is also crucial for intact protein analysis.
Sample complexity must be minimized if intact protein analysis by MS is to be successful. Proteins must be well-resolved from one another and from various proteoforms to minimize the low thresholds of intact protein detection. Thermo Scientific ProSwift RP-4H LC Columns are ideal for use in intact protein separation prior to MS analysis; they are also designed to work with milliliter to microliter flow.
Designed for ultra high-resolution separation of intact proteins, the Thermo Scientific UltiMate 3000 Rapid Separation systems, including the Thermo Scientific UltiMate 3000 RSLCnano System with featured Thermo Scientific ProFlow technology, enable milliliter to nanoliter flow separations and easy integration with mass spectrometry analyzers, providing the best resolution, sensitivity, and selectivity for intact protein applications.
Intact protein analysis relies on the ultra HRAM capacity of the Thermo Scientific Orbitrap Fusion Lumos Tribrid Mass Spectrometer. This system offers the highest resolving power of all Orbitrap-based instruments (up to 500,000 at m/z 200); likewise, its unique Advanced Vacuum Technology increases ion transmission and improves the quality of acquired intact protein mass spectra. Such advantages facilitate efficient enrichment of individual isobaric protein forms and subsequent top-down analysis.
The Thermo Scientific Q Exactive HF Hybrid Quadrupole-Orbitrap Mass Spectrometer, with its optional Intact Protein Mode, enables high-resolution analysis of complex intact proteins and permits detailed characterization of larger and highly charged species such as entire antibodies and their individual light and heavy chains. With the additional resolving power of the Q Exactive HF MS, routine yet detailed characterization of proteins and biopharmaceuticals up to 50 kDa is possible.
Facilitate analysis of top-down and middle-down proteomics data with Thermo Scientific ProSightPC Software, a stand-alone software platform that also characterizes protein PTMs. ProsightPC software not only identifies intact proteins and protein digests using high-resolution, accurate-mass MS and MS/MS data, it also enables the user to search those data against proteome repositories such as UniProt.