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Introduction to Mass Spectrometry for Biomolecules

Mass spectrometry (MS) plays an important role in the analysis and characterization of protein biopharmaceuticals.  It is utilized at almost every stage of development, including clone selection, cell-culture process development, purification process development, formulation development, stability studies, comparability studies, structural characterization and quality control.  

Protein features that can be characterized by MS include accurate molecular weight, amino acid sequence, N- and C-termini, disulfide linkages, glycan structure and profile, co- and post-translational modifications and modification sites, drug-to-antibody ratio (DAR), drug load distribution and conjugation sites, sequence variants, higher order structures etc.1

These analyses require high-resolution accurate mass instruments with MS/MS capabilities, traditionally these would have been quadrupole time-of-flight (Q-ToF) instruments, however, advances in MS technology have resulted in the increased use of hybrid Orbitrap MS systems.  

The majority of MS applications use electrospray ionization (ESI) which is normally performed using denaturing conditions (organic solvents and acids); however, there has been increased interest in native ESI which utilizes volatile buffers at neutral pH.  Matrix assisted laser desorption ionization (MALDI) has also been applied to the study of protein biopharmaceuticals, but suffers from poor mass accuracy and resolution for larger molecules.  This is a direct result of the limited charging of molecules under this mode of ionization, and therefore requires the use of ToF mass analyzers in low-resolution linear mode to capture the m/z values generated by proteins.  

A wide range of fragmentation techniques are used for MS/MS experiments with proteins, peptides, and glycans; collision-induced dissociation (CID) is most commonly used, however, alternatives such as higher-energy collision dissociation (HCD), electron capture dissociation (ECD) and electron transfer dissociation (ETD) can provide additional structural information (Figure 1).

Figure 1: Fragmentation patterns from CID, HCD, ETD (fragment z’), and ECD (fragment c).

In biopharmaceutical analysis, MS is often coupled with a variety of chromatographic and electrophoretic techniques, either on- or offline depending on the technique.  Reversed phase HPLC and HILIC are directly compatible with MS, whereas hydrophobic interaction (HIC), ion exchange (IEX), and size exclusion (SEC) chromatography are not directly compatible with MS due to the use of involatile salts, and so require desalting or the use of 2D-LC.  This being said, many manufacturers offer solutions for desalting the chromatographic eluent prior to introduction into the MS and as such the coupling of these other techniques with MS detection is possible.   

MS determination can be carried out on intact or fragmented proteins and are categorized as intact, middle-up, bottom-up, top-down, and middle-down depending on the type of analysis and fragments being analyzed.  MS assessment of intact proteins provides information about the protein as a whole, including in some cases tertiary and quaternary structure, but will not afford information relating to types or locations of any modifications. 

To gather this type of information the protein must be digested into smaller fragments, if carried out in solution using enzymes or reducing agents, these fragments can then be analyzed using a mass spectrometer; known as an up-type method.  Down-type methods analyze the entire protein directly by fragmenting it in the gas phase inside the mass spectrometer.  The nomenclature of intact, top, middle, and bottom relate to the size of the molecule being introduced into the mass spectrometer in relation to the intact molecule (Figure 2).  

Each technique has its own advantages and disadvantages in terms of structural information provided, sequence coverage, sample consumption, and ease of analysis.  Often full protein characterizations will be carried out using multiple methods due to their complimentary nature.

Figure 2: Digestion and cleavage pathways.

 

 

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