Maximizing Selective Cleavages at Aspartic Acid and Proline Residues for the Identification of Intact Proteins

Abstract

A new approach for the identification of intact proteins has been developed that relies on the generation of relatively few abundant products from specific cleavage sites. This strategy is intended to complement standard approaches that seek to generate many fragments relatively non-selectively. Specifically, this strategy seeks to maximize selective cleavage at aspartic acid and proline residues via collisional activation of precursor ions formed via electrospray ionization (ESI) under denaturing conditions. A statistical analysis of the SWISS-PROT database was used to predict the number of arginine residues for a given intact protein mass and predict a m/z range where the protein carries a similar charge to the number of arginine residues thereby enhancing cleavage at aspartic acid residues by limiting proton mobility. Cleavage at aspartic acid residues is predicted to be most favorable in the m/z range of 1500-2500, a range higher than that normally generated by ESI at low pH. Gas-phase proton transfer ion/ion reactions are therefore used for precursor ion concentration from relatively high charge states followed by ion isolation and subsequent generation of precursor ions within the optimal m/z range via a second proton transfer reaction step. It is shown that the majority of product ion abundance is concentrated into cleavages C-terminal to aspartic acid residues and N-terminal to proline residues for ions generated by this process. Implementation of a scoring system that weights both ion fragment type and ion fragment area demonstrated identification of standard proteins, ranging in mass from 8.5 to 29.0 kDa. Graphical Abstract ᅟ.

Origin and Prediction of Highly Specific Bond Cleavage Sites in the Thermal Activation of Intact Protein Ions

“…[21a] At higher charges tates, additional dominant cleavage sites are observed. For example, CID of the [ubiquitin, 16 H] 16 + resultsi n three very abundantc leavage sites between residues Ile 13 -Pro 19 ,S er 20 -Asp 21 and Leu 56 -Gln 62 ( Figure 1L). In addition, specific cleavages C-terminal to Asp residues are observed for low charges tates ( 12 +)o fu biquitin,w hich is known as the "aspartic acid" effect (refer to Supporting Information for details).…”

“…Others have investigated the effects of charge states on the CID of protein ions . Unlike for peptides, the CID of protein ions in low charge states results in relatively extensive fragmentation and non‐selective fragmentation, and cleavage sites are often identified N‐terminal to Pro and C‐terminal to Asp and Glu . Also in contrast to peptides, the CID of proteins in relatively high charge states results in an exceedingly limited number of dominant backbone cleavage sites.…”

Ion-Ion Proton Transfer and Parallel Ion Parking for the Analysis of Mixtures of Intact Proteins on a Modified Orbitrap Mass Analyzer

“…Co-elution of multiple proteoforms and distribution of ion signal for each proteoform among multiple charge states—each having multiple isotopic m/z peaks—leads to the low S:N inherent to the currently utilized approaches for analysis of these large biological molecules. It is clear from the recent efforts of the Brodbelt and McLuckey laboratories [25, 26] that ion parking and parallel ion parking techniques are potentially advantageous in this challenging MS application area. We believe that the combination of IIPT and parallel ion parking with reagent activation (PIP-RA) will, with further improvements in the methodology and accompanying improvements to MS instrumentation, become a powerful tool in the study of intact protein mixtures.…”

Identification and Quantification of Proteoforms by Mass Spectrometry

“…Therefore, it is common for fragment ion signals to fall below detection limits. Techniques such as charge‐reduction and ion‐parking have been used to simplify spectra and enhance sensitivity. However, sensitivity for identification of proteoforms diminishes severely beyond 30 kDa …”