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The NISTmAb Tryptic Peptide Spectral Library for Monoclonal Antibody Characterization



Qian Dong, Yuxue Liang, Xinjian Yan, Sanford Markey, Yuri Mirokhin, Dmitrii V. Tchekhovskoi, Tallat H. Bukhari, Stephen E. Stein


We describe the creation of a mass spectral library composed of all identifiable spectra derived from the tryptic digest of a therapeutic monoclonal antibody. The NISTmAb library is a unique reference peptide spectral library developed from a total of six million MS2 spectra acquired by nanospray ESI LC-MS/MS over a wide range of HCD collision energies with both conventional 1D separation for various digestion conditions and 20-24 fraction 2D-LC studies of a single digest. Computer methods were developed for automated analysis of LC-MS isotopic clusters to determine the MS1 attributes for all ions detected in the 1D and 2D studies. The library contains 12,611 high quality HCD spectra of 3,361 peptide ions identified and validated by accurate mass, differential elution pattern, and expected peptide classes in peptide map experiments. These include a variety of biologically modified peptide spectra involving glycosylated, oxidized, deamidated, N/C-terminal variants, glycated peptides, and protein clips as well as artifactual peptides. A complete glycation profile was obtained for NISTmAb with spectra for 63 % and 100 % of possible glycation sites in the heavy and light chains, respectively. The site-specific quantification of methionine oxidation in the protein is described. The utility of this reference library is demonstrated by the analysis of a commercial mAb (Humira), where 691 peptides are identified in the constant regions, accounting for 60 % coverage for both heavy and light chains, including many low-abundance modified peptides. The NIST reference library platform may be used as a tool for facile identification of primary sequences and their PTMs, as well as the recognition of method-induced artifacts in the constant regions for many human- IgGs and recombinant antibodies. Its development also provides a general method for creating comprehensive peptide libraries of individual proteins.


NISTmAb, tryptic peptide spectral library, monoclonal antibody, HCD fragmentation, post-translational modifications, quantitation, 1D/2D-LC MS/MS


Dong, Q. , Liang, Y. , Yan, X. , Markey, S. , Mirokhin, Y. , Tchekhovskoi, D. , Bukhari, T. and Stein, S. (2018), The NISTmAb Tryptic Peptide Spectral Library for Monoclonal Antibody Characterization, mAbs, [online],, (Accessed May 19, 2024)


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Created March 6, 2018, Updated October 13, 2022