Pepsin-Containing Membranes for Controlled Monoclonal Antibody Digestion Prior to Mass Spectrometry Analysis

Abstract

Monoclonal antibodies (mAbs) are the fastest growing class of therapeutic drugs, because of their high specificities to target cells. Facile analysis of therapeutic mAbs and their post-translational modifications (PTMs) is essential for quality control, and mass spectrometry (MS) is the most powerful tool for antibody characterization. This study uses pepsin-containing nylon membranes as controlled proteolysis reactors for mAb digestion prior to ultrahigh-resolution Orbitrap MS analysis. Variation of the residence times (from 3 ms to 3 s) of antibody solutions in the membranes yields “bottom-up” (1–2 kDa) to “middle-down” (5–15 kDa) peptide sizes within less than 10 min. These peptides cover the entire sequences of Trastuzumab and a Waters antibody, and a proteolytic peptide comprised of 140 amino acids from the Waters antibody contains all three complementarity determining regions on the light chain. This work compares the performance of “bottom-up” (in-solution tryptic digestion), “top-down” (intact protein fragmentation), and “middle-down” (in-membrane digestion) analysis of an antibody light chain. Data from tandem MS show 99%, 55%, and 99% bond cleavage for “bottom-up”, “top-down”, and “middle-down” analyses, respectively. In-membrane digestion also facilitates detection of PTMs such as oxidation, deamidation, N-terminal pyroglutamic acid formation, and glycosylation. Compared to “bottom-up” and “top-down” approaches for antibody characterization, in-membrane digestion uses minimal sample preparation time, and this technique also yields high peptide and sequence coverage for the identification of PTMs.

Higher-order structural interrogation of antibodies using middle-down hydrogen/deuterium exchange mass spectrometry

“…During the revision process of the current paper, an interesting study published which used pepsin-containing nylon membranes for controlled proteolysis of reduced antibodies, but it was not used for HDX studies. 25 Direct ETD/ECD analysis of intact antibodies normally can only provide information for non-disulfide-linked regions. 26 , 27 The digestion condition required by HDX experiments (pH 2.5) can lead to protein denaturation, which may result in non-specific cleavage.…”

Solvent composition dependent signal reduction of molecular ions generated from aromatic compounds in (+) atmospheric pressure photoionization mass spectrometry

“…Liquid chromatography coupled with mass spectrometry (LC/MS) is one of the most important analytical tools in contemporary scientific and industrial research on food, 1-3 drugs, 4,5 petroleum, [6][7][8] biomolecules, [9][10][11][12][13][14][15][16][17][18][19][20] and environmental samples. 21,22 For this reason, a considerable amount of effort has been devoted to improving the analytical sensitivity of LC/MS, [23][24][25][26] and it is well known that ionization is a critical step that can greatly influence the analytical sensitivity of LC/MS.…”

Time, space, and disorder in the expanding proteome universe

“…These highly acidic conditions would be only necessary after conformational features are “encoded” as deuterium incorporation and thus do not affect the native structural states of cells. Ultra‐rapid digestion methods, mass‐spectrometry compatible detergents and faster computation of complex spectra resulting from a large number of variable isotope changes may further help to pave the way toward proteome‐wide in vivo HDX experiments .…”