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

Abstract

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 (m/z-value 16,950) and ribonuclease B (m/z-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 m/z-value 12,000 (e.g., resolving power 39,000 at m/z-value 12,000) providing more reliable identifications. Moreover, examples are presented of pseudo-MS(3) experiments on ISD fragment ions from RNase B by collisional-induced dissociation (CID).

In-Source Decay and Pseudo-MS3of Peptide and Protein Ions Using Liquid AP-MALDI

“…The experiments were performed on a Q-Exactive Plus equipped with an AP-MALDI imaging source, which thus enabled accurate mass, high mass resolution characterization of the ISD, and pseudo-MS 3 fragments. Thus, the method extends the high mass resolution, accurate mass ISD recently reported by Nicolardi et al [8] to also include targeted ISD fragment sequencing and optimized liquid matrices.…”

“…The major drawbacks of ISD are the lack of precursor ion selection and the matrix cluster background that can obscure fragment ions in the lower mass range. The ultra-high mass resolution and high mass accuracy of Fourier transform mass spectrometry have been exploited to resolve more of the fragments [8]. Alternatively, a pseudo-MS 3 method incorporating ISD-fragment-isolation and post-source decay (PSD) has been used to acquire sequence tags from specific ISD fragments, and therefore unencumbered by the matrix background.…”

Top-down proteomics: applications, recent developments and perspectives

“…It was reported that using top-down MALDI-FTICR-MS platform, 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 [145]. It was proved that mass spectrometry (MS)-based top-down proteomics, multi-dimensional liquid chromatography (MDLC) strategies can effectively separate intact proteins with high resolution and automation are highly desirable [36].…”

In-source decay characterization of isoaspartate and β-peptides

“…ISD has been applied to analyze peptides and proteins with phosphorylation and O - and N -glycosylation, to differentiate the isomeric amino acid residues leucine and isoleucine, and to obtain information on disulfide-linkages. 50–55 Despite early speculation that ISD may result from electron capture by the multiply-charged molecular species in the MALDI plume, 56 it is now generally accepted that ISD is initiated by hydrogen transfer from the matrix molecule to the backbone carbonyl oxygen. 48 A number of matrices have been tested for ISD experiments, including picolinic acid (PA), 1,5-diaminonaphthalene (1,5-DAN), and 2,5-dihydroxybenzoic acid (DHB).…”