Top-Down MALDI-In-Source Decay-FTICR Mass Spectrometry of Isotopically Resolved Proteins

An accurate mass measurement of a known protein provides information on potential amino acid deletions and post-translational modifications. Although this field is dominated by strategies based on electrospray ionization, mass spectrometry (MS) methods using matrix-assisted laser desorption/ionization (MALDI) have the advantage of yielding predominantly singly charged precursor ions, thus avoiding peak overlap from different charge states of multiple species. Such MALDI-MS methods require mass measurement at ultrahigh resolution, which is provided by Fourier transform ion cyclotron resonance (FTICR) mass analyzers. Recently, using a MALDI-FTICR-MS platform equipped with a 15 T magnet, we reported on the mass analysis of intact human serum peptides and small proteins with isotopic resolution up to ∼15 kDa and identified new proteoforms from an accurate measurement of mass distances. In the current study, we have used this FTICR system after an upgrade with a novel dynamically harmonized ICR cell, i.e., ParaCell, for mapping isotopically resolved intact proteins up to about 17 kDa and performed top-down MALDI in-source decay (ISD) analysis. Standard proteins myoglobin (<i>m</i>/<i>z</i>-value 16 950) and ribonuclease B (<i>m</i>/<i>z</i>-value 14 900) were measured with resolving powers of 62 000 and 61 000, respectively. Furthermore, it will be shown that (singly charged) MALDI-ISD fragment ions can be measured at isotopic resolution up to <i>m</i>/<i>z</i>-value 12 000 (e.g., resolving power 39 000 at <i>m</i>/<i>z</i>-value 12 000) providing more reliable identifications. Moreover, examples are presented of pseudo-MS³ experiments on ISD fragment ions from RNase B by collisional-induced dissociation (CID)