Validation of Top-Down Proteomics Data by Bottom-Up-Based N-Terminomics Reveals Pitfalls in Top-Down-Based Terminomics Workflows

Abstract

Bottom-up proteomics (BUP)-based N-terminomics techniques have become standard to identify protein N-termini. While these methods rely on the identification of N-terminal peptides only, top-down proteomics (TDP) comes with the promise to provide additional information about post-translational modifications and the respective C-termini. To evaluate the potential of TDP for terminomics, two established TDP workflows were employed for the proteome analysis of the nematode Caenorhabditis elegans. The N-termini of the identified proteoforms were validated using a BUP-based N-terminomics approach. The TDP workflows used here identified 1658 proteoforms, the N-termini of which were verified by BUP in 25% of entities only. Caveats in both the BUP- and TDP-based workflows were shown to contribute to this low overlap. In BUP, the use of trypsin prohibits the detection of arginine-rich or arginine-deficient N-termini, while in TDP, the formation of artificially generated termini was observed in particular in a workflow encompassing sample treatment with high acid concentrations. Furthermore, we demonstrate the applicability of reductive dimethylation in TDP to confirm biological N-termini. Overall, our study shows not only the potential but also current limitations of TDP for terminomics studies and also presents suggestions for future developments, for example, for data quality control, allowing improvement of the detection of protein termini by TDP.

Mass Spectrometry Analysis on the Breakage of Allergens in High-Molecular-Mass Polymer of Roasted Peanuts

“…Structure of peptide in protein after broken. The peptide RQQWELQGDRRC is a fragment of Ara h 2 in roasted peanuts. (A, B) Original structure.…”

“…Dimethylation can cause mass increments of 32 Da on the N-termini of proteins, 32 and labeling before digestion partly avoids the disturbance of trypsin peptides. 33,34 As shown in Table 1, we detected five N-termini with dimethyl-labeled (shown in Figures 2A and S1), including the N-termini of mature Ara h 2 (R22, No. 12 in Table 1) and Ara h 3 (I21, No.…”

“…56,57 Roasting might affect the conformation of epitopes. The structure of breakage peptide R( 22)QQWELQGDRRC (33) was simulated as shown in Figure 5, which refers to the breakage site No. 13 in Table 1 and the epitopes No.…”

Identification of proteoforms by top‐down proteomics using two‐dimensional low/low pH reversed‐phase liquid chromatography‐mass spectrometry

“…Therefore, we concluded that avoidance of heating and prolonged exposure to acidic conditions is necessary but also sufficient to prevent artificial cleavage of highly labile peptide bonds. Nevertheless, further studies are needed to clarify the origin of these subsequence proteoforms, that is, if they arise mainly biologically [38][39][40] or artificially, for example, by applying N-terminal labeling strategies [41].…”

Proteoforms expand the world of microproteins and short open reading frame-encoded peptides

“… 63 Reductive dimethylation has also been shown to help evaluating N-termini in proteoforms identified by TDP-based terminomics. 63 , 64 …”