Effective Amino Acid Sequencing of Intact Filgrastim by Multimodal Mass Spectrometry with Topdownr

Abstract

Therapeutic proteins, known as biologicals, are an important and growing class of drugs for treatment of a series of human ailments. Amino acid sequence variants of therapeutic proteins can affect their safety and efficacy. Top-down mass spectrometry is well suited for the sequence analysis of intact therapeutic proteins. Fine-tuning of tandem mass spectrometry (MS/ MS) fragmentation conditions is essential for maximizing the amino acid sequence coverage but is often time-consuming. We used topdownr, an automated and integrated multimodal approach to systematically assess high mass accuracy MS/MS fragmentation parameters to characterize filgrastim, a 19 kDa recombinant human granulocyte colony-stimulating factor used in treating neutropenia. A total of 276 different MS/MS conditions were systematically tested, including the following parameters: protein charge state, HCD and CID collision energy, ETD reaction time, ETD supplemental activation, and UVPD activation time. Stringent and accurate evaluation and annotation of the MS/MS data was achieved by requiring a fragment ion mass error of 5 ppm, considering reproducible N-and C-terminal fragment ions only, and excluding internal fragment ion assignments. We report the first EThcD and UVPD MS/MS analysis of intact filgrastim, and these two techniques combined resulted in 98% amino acid sequence coverage. By combining all tested fragmentation modes, we obtained near-complete amino acid sequence coverage (99.4%) of intact filgrastim.

How to deal with internal fragment ions?

“…For each protein, 108 unique fragmentation settings were used. For the filgrastim analysis protocol refer to Babovic et al [12]. The specific fragmentation method and parameters of MSMS spectra in the analysis are specified in the figure captions.…”

“…Top-down or middle-down MS experiments are sometimes integrated with data from bottom-up experiments to capitalize on both approaches’ advantages [9, 10, 11]. Insufficient sequence information in top-down approaches has also been pondered, either by integrating multiple MS2 fragmentation techniques to enhance sequence coverage [12] or by performing multiple levels of fragmentation of a protein ion, known as MSn [13, 14]. A drawback of these methods is that they involve several rounds of analysis to be carried out and therefore require larger sample amounts.…”