TY - JOUR
T1 - Not All Arms of IgM Are Equal
T2 - Following Hinge-Directed Cleavage by Online Native SEC-Orbitrap-Based CDMS
AU - Yin, Victor
AU - Deslignière, Evolène
AU - Mokiem, Nadia
AU - Gazi, Inge
AU - Lood, Rolf
AU - de Haas, Carla J.C.
AU - Rooijakkers, Suzan H.M.
AU - Heck, Albert J.R.
N1 - Publisher Copyright:
© 2024 The Authors. Published by American Chemical Society.
PY - 2024/6/5
Y1 - 2024/6/5
N2 - Immunoglobulins M (IgM) are key natural antibodies produced initially in humoral immune response. Due to their large molecular weights and extensive glycosylation loads, IgMs represent a challenging target for conventional mass analysis. Charge detection mass spectrometry (CDMS) may provide a unique approach to tackle heterogeneous IgM assemblies, although this technique can be quite laborious and technically challenging. Here, we describe the use of online size exclusion chromatography (SEC) to automate buffer exchange and sample introduction, and demonstrate its adaptability with Orbitrap-based CDMS. We discuss optimal experimental parameters for online SEC-CDMS experiments, including ion activation, choice of column, and resolution. Using this approach, CDMS histograms containing hundreds of individual ion signals can be obtained in as little as 5 min from single injections of <1 μg of sample. To demonstrate the unique utility of online SEC-CDMS, we performed real-time kinetic monitoring of pentameric IgM digestion by the protease IgMBRAZOR, which cleaves specifically in the hinge region of IgM. Several digestion intermediates corresponding to processive losses of F(ab’)2 subunits could be mass-resolved and identified by SEC-CDMS. Interestingly, we find that for the J-chain linked IgM pentamer, cleavage of one of the F(ab’)2 subunits is much slower than the other four F(ab’)2 subunits, which we attribute to the symmetry-breaking interactions of the J-chain within the pentameric IgM structure. The online SEC-CDMS methodologies described here open new avenues into the higher throughput automated analysis of heterogeneous, high-mass protein assemblies by CDMS.
AB - Immunoglobulins M (IgM) are key natural antibodies produced initially in humoral immune response. Due to their large molecular weights and extensive glycosylation loads, IgMs represent a challenging target for conventional mass analysis. Charge detection mass spectrometry (CDMS) may provide a unique approach to tackle heterogeneous IgM assemblies, although this technique can be quite laborious and technically challenging. Here, we describe the use of online size exclusion chromatography (SEC) to automate buffer exchange and sample introduction, and demonstrate its adaptability with Orbitrap-based CDMS. We discuss optimal experimental parameters for online SEC-CDMS experiments, including ion activation, choice of column, and resolution. Using this approach, CDMS histograms containing hundreds of individual ion signals can be obtained in as little as 5 min from single injections of <1 μg of sample. To demonstrate the unique utility of online SEC-CDMS, we performed real-time kinetic monitoring of pentameric IgM digestion by the protease IgMBRAZOR, which cleaves specifically in the hinge region of IgM. Several digestion intermediates corresponding to processive losses of F(ab’)2 subunits could be mass-resolved and identified by SEC-CDMS. Interestingly, we find that for the J-chain linked IgM pentamer, cleavage of one of the F(ab’)2 subunits is much slower than the other four F(ab’)2 subunits, which we attribute to the symmetry-breaking interactions of the J-chain within the pentameric IgM structure. The online SEC-CDMS methodologies described here open new avenues into the higher throughput automated analysis of heterogeneous, high-mass protein assemblies by CDMS.
UR - http://www.scopus.com/inward/record.url?scp=85194045928&partnerID=8YFLogxK
U2 - 10.1021/jasms.4c00094
DO - 10.1021/jasms.4c00094
M3 - Article
C2 - 38767111
AN - SCOPUS:85194045928
SN - 1044-0305
VL - 35
SP - 1320
EP - 1329
JO - Journal of the American Society for Mass Spectrometry
JF - Journal of the American Society for Mass Spectrometry
IS - 6
ER -