Improving data quality and preserving HCD-generated reporter ions with EThcD for isobaric tag-based quantitative proteomics and proteome-wide PTM studies

Abstract

Mass spectrometry (MS)-based isobaric labeling has undergone rapid development in recent years due to its capability for high throughput quantitation. Apart from its originally designed use with collision-induced dissociation (CID) and higher-energy collisional dissociation (HCD), isobaric tagging technique could also work with electron transfer dissociation (ETD), which provides complementarity to CID and is preferred in sequencing peptides with post-translational modifications (PTMs). However, ETD suffers from long reaction time, reduced duty cycle and bias against peptides with lower charge states. In addition, common fragmentation mechanism in ETD results in altered reporter ion production, decreased multiplexing capability, and even loss of quantitation capability for some of the isobaric tags, including custom-designed dimethyl leucine (DiLeu) tags. Here, we demonstrate a novel electron-transfer/higher-energy collision dissociation (EThcD) approach that preserves original reporter ion channels, mitigates bias against lower charge states, improves sensitivity, and significantly improves data quality for quantitative proteomics and proteome-wide PTM studies. Systematic optimization was performed to achieve a balance between data quality and sensitivity. We provide direct comparison of EThcD with ETD and HCD for DiLeu- and TMT-labeled HEK cell lysate and IMAC enriched phosphopeptides. Results demonstrate improved data quality and phosphorylation localization accuracy while preserving sufficient reporter ion production. Biological studies were performed to investigate phosphorylation changes in a mouse vascular smooth muscle cell line treated with four different conditions. Overall, EThcD exhibits superior performance compared to conventional ETD and offers distinct advantages compared to HCD in isobaric labeling based quantitative proteomics and quantitative PTM studies.

Site-specific characterization and quantitation ofN-glycopeptides in PKM2 knockout breast cancer cells using DiLeu isobaric tags enabled by electron-transfer/higher-energy collision dissociation (EThcD)

“…56–61 Recently, our group has successfully applied DiLeu tags for quantitation of labile PTM enabled by EThcD. 62 Even though ETD has been well-known to preserve labile PTMs, the intensity of reporter ions produced is often quite low so that the quantitation accuracy is compromised. While HCD provides high reporter ion intensity, labile PTMs are poorly preserved.…”

“…Utilizing the novel EThcD hybrid fragmentation method, where a supplemental energy is applied to all fragment ions formed by ETD, we were able to successfully apply DiLeu tags for reliable quantitative phosphoproteomics study with increased phosphorylation site confidence. 62 In this regard, we propose quantitation of intact glycopeptides could also benefit from EThcD for site-specific characterization as well as accurate quantitation. As shown in Figure 4A, reporter ions with decent intensities next to the base peak (HexNAc oxonium ion) were produced in the low mass region, which facilitated the accurate and reliable quantitation of intact glycopeptides.…”

Acetyl-CoA flux from the cytosol to the ER regulates engagement and quality of the secretory pathway

“…custom-packed BEH C18 (1.7 μm, 130 Å, Waters) capillary column with an 80 min gradient from 0 to 30% ACN (0.1% FA). Mass spectrometer was operated in a top 20 data-dependent acquisition mode with HCD-product dependent-EThcD fragmentation 44 . Survey scans of peptide precursors from m/z 400 to 2000 were performed at resolving power of 120 K and AGC target of 4 × 10 5 with a maximum injection time of 150 ms. Tandem MS acquisition was at resolving power of 60 K, AGC target of 5 × 10 4 and dynamic exclusion of 12 s of 10 ppm mass tolerance.…”

Mass Spectrometry-based Proteomics and Glycoproteomics in COVID-19 Biomarkers Identification: A Mini-review

“…The different choices of peptide fragmentation include higher-energy collisional dissociation (HCD), collision-induced dissociation (CID), or electron-transfer dissociation (ETD) which are well covered in other reviews and hence will not be discussed here [ 66 , 67 ]. What is worth mentioning is that ETD performs the ionization by transferring electrons to a multiply protonated peptide so is widely used to identify PTMs on the peptide, and in certain cases will be combined with HCD to identify modifications [ 68 ]. The obtained raw data will then be analyzed to acquire peptide sequence and quantitation in software, such as MaxQuant, Mascot, etc.…”