Improved N- and C-Terminal Sequencing of Proteins by Combining Positive and Negative Ion MALDI In-Source Decay Mass Spectrometry

Abstract

The development of various ionization and fragmentation techniques has been of key importance for establishing mass spectrometry (MS) as a powerful tool for protein characterization. One example of this is matrix-assisted laser desorption/ionization (MALDI) combined with in-source decay (ISD) fragmentation that allows mapping of N- and C-terminal regions of large proteins without the need for proteolysis. Positive ion mode ISD fragments are commonly assigned in the mass region above m / z 1000, while MALDI matrix ions generally hamper the detection of smaller singly charged fragments. The ultrahigh resolving power provided by Fourier transform ion cyclotron resonance (FT-ICR) MS partially overcomes this limitation, but to further increase the detection of smaller fragments we have revisited the application of negative ion mode MALDI-ISD and found good coverage of the peptide chain termini starting from c′2 and z′2 fragment ions. For the first time, we demonstrate that the combination of negative and positive ion MALDI FT-ICR MS is a useful tool to improve the characterization of mAbs. The different specificities of the two ion modes allowed us to selectively cover the sequence of the light and heavy chains of mAbs at increased sensitivity. A comprehensive evaluation of positive and negative ion mode MALDI-ISD FT-ICR MS in the m / z range 46–13 500 showed an increased sequence coverage for three standard proteins, namely, myoglobin, SiLuLite mAb, and NIST mAb. The data obtained in the two ion modes were, in part, complementary.

Protein Characterization by MALDI In-Source Decay Mass Spectrometry in Support of Safety Assessments of Genetically Modified Crops

“…30 Others used Fourier transform ion cyclotron resonance MS in the positive and negative modes to extend the ISD accessible range in both directions to m/z 46− 13,500. 31 T 3 Sequencing. During ISD, the multitude of intense peaks of MALDI matrix ions overwhelms the spectra in the m/ z 0−800 region, making it nearly impossible to determine the first 5−8 amino acids of the N-and C-termini.…”

“…22,37 At the same time, the presence of acidic amino acids or acidic post-translational modifications in the sequence while potentially decreasing the ability to detect ISD fragments in the positive mode may provide necessary sequence information through the analysis of negatively charged ISD fragments. 29,31 The MALDI ISD spectrum of microbially produced AvHPPD-03 collected in the positive mode includes a long series of c fragment ions which almost completely covers the N-terminal sequence of AvHPPD-03 from Gly9 to Glu46 (data not shown). This portion of the protein contains 5 Arg and 3 His residues and only 2 Glu and 1 Asp, which ensures that the N-terminal ISD fragments will be detected in the positive mode.…”

“…For example, Resemann and co-workers completely sequenced the subunits of immunoglobulins cetuximab, panitumumab, and natalizumab using MALDI ISD by extending the coverage limits to approximately m / z 15,000 . Others used Fourier transform ion cyclotron resonance MS in the positive and negative modes to extend the ISD accessible range in both directions to m / z 46–13,500 …”

“…The observation of ISD fragments is the result of two independent processes occurring during the MALDI assessment: (1) formation of N- and C-terminal fragments of proteins or peptides mostly via radical-driven fragmentation and (2) ionization of resultant fragments to form protonated positively charged [M + H] + or deprotonated negatively charged [M – H] − species . The majority of studies using MALDI ISD of proteins and peptides are conducted in a positive mode, and the ionization efficiency of ISD fragment ions strongly depends on the presence of basic amino acids such as Arg, Lys, and His, with Arg being the most efficient. , At the same time, the presence of acidic amino acids or acidic post-translational modifications in the sequence while potentially decreasing the ability to detect ISD fragments in the positive mode may provide necessary sequence information through the analysis of negatively charged ISD fragments. , …”

Native Liquid Chromatography and Mass Spectrometry to Structurally and Functionally Characterize Endo-Xylanase Proteoforms

“…The MALDI−insource decay (ISD) FT−ICR MS was performed as previously reported [39][40][41]. In short, the SEC fractions (ENDO-I and glucoamylase) and IEX fractions (mature and +222 Da peak) were collected, and buffer exchanged using 3 kDa Vivaspin 2 filters to 5% MeOH or 50 mM ammonium formate, respectively.…”