The Consortium for Top-Down Proteomics (www.topdownproteomics.org) launched the present study to assess the current state of top-down mass spectrometry (TD MS) and middle-down mass spectrometry (MD MS) for characterizing monoclonal antibody (mAb) primary structures, including their modifications. To meet the needs of the rapidly growing therapeutic antibody market, it is important to develop analytical strategies to characterize the heterogeneity of a therapeutic product's primary structure accurately and reproducibly. The major objective of the present study is to determine whether current TD/MD MS technologies and protocols can add value to the more commonly employed bottom-up (BU) approaches with regard to confirming protein integrity, sequencing variable domains, avoiding artifacts, and revealing modifications and their locations. We also aim to gather information
Accurate sequence
characterization is essential for the development
of therapeutic antibodies by the pharmaceutical industry. Presented
here is a methodology to obtain comprehensive sequence analysis of
a monoclonal antibody. An enzyme reactor of immobilized Aspergillopepsin
I, a highly stable nonspecific protease, was used to cleave reduced
antibody subunits into a peptide profile ranging from 1 to 20 kDa.
Utilizing the Thermo Orbitrap Fusion’s unique instrument architecture
combined with state-of-the-art instrument control software allowed
for dynamic instrument methods that optimally characterize eluting
peptides based on their size and charge density. Using a data-dependent
instrument method, both collisional dissociation and electron transfer
dissociation were used to fragment the appropriate charge state of
analyte peptides. The instrument layout also allowed for scans to
be taken in parallel using both the ion trap and Orbitrap concurrently,
thus allowing larger peptides to be analyzed in high resolution using
the Orbitrap while simultaneously analyzing tryptic-like peptides
using the ion trap. We harnessed these capabilities to develop a custom
method to optimally fragment the eluting peptides based on their mass
and charge density. Using this approach, we obtained 100% sequence
coverage of the total antibody in a single chromatographic analysis,
enabling unambiguous sequence assignment of all residues.
Membrane proteins are challenging to analyze by native mass spectrometry (MS) as their hydrophobic nature typically requires stabilization in detergent micelles that are removed prior to analysis via collisional activation. There is however a practical limit to the amount of energy which can be applied, which often precludes subsequent characterization by top‐down MS. To overcome this barrier, we have applied a modified Orbitrap Eclipse Tribrid mass spectrometer coupled to an infrared laser within a high‐pressure linear ion trap. We show how tuning the intensity and time of incident photons enables liberation of membrane proteins from detergent micelles. Specifically, we relate the ease of micelle removal to the infrared absorption of detergents in both condensed and gas phases. Top‐down MS via infrared multiphoton dissociation (IRMPD), results in good sequence coverage enabling unambiguous identification of membrane proteins and their complexes. By contrasting and comparing the fragmentation patterns of the ammonia channel with two class A GPCRs, we identify successive cleavage of adjacent amino acids within transmembrane domains. Using gas‐phase molecular dynamics simulations, we show that areas prone to fragmentation maintain aspects of protein structure at increasing temperatures. Altogether, we propose a rationale to explain why and where in the protein fragment ions are generated.
Membrane proteins are challenging to analyze by native mass spectrometry (MS) as their hydrophobic nature typically requires stabilization in detergent micelles that are removed prior to analysis via collisional activation. There is however a practical limit to the amount of energy which can be applied, which often precludes subsequent characterization by top‐down MS. To overcome this barrier, we have applied a modified Orbitrap Eclipse Tribrid mass spectrometer coupled to an infrared laser within a high‐pressure linear ion trap. We show how tuning the intensity and time of incident photons enables liberation of membrane proteins from detergent micelles. Specifically, we relate the ease of micelle removal to the infrared absorption of detergents in both condensed and gas phases. Top‐down MS via infrared multiphoton dissociation (IRMPD), results in good sequence coverage enabling unambiguous identification of membrane proteins and their complexes. By contrasting and comparing the fragmentation patterns of the ammonia channel with two class A GPCRs, we identify successive cleavage of adjacent amino acids within transmembrane domains. Using gas‐phase molecular dynamics simulations, we show that areas prone to fragmentation maintain aspects of protein structure at increasing temperatures. Altogether, we propose a rationale to explain why and where in the protein fragment ions are generated.
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