PTM Hotspot

1 Introduction

PTM Hotspot is a tool designed to identify potential post-translational modification (PTM) risk sites within antibody sequences. By scanning sequences, it accurately locates PTM sites that may affect antibody stability, activity, or immunogenicity, providing critical data support for antibody drug development. Additionally, PTM Hotspot features powerful visualization capabilities, presenting PTM risk site information in an intuitive sequence map. It also enables users to visually assess, via scatter plots, whether sequences fall within therapeutic antibody risk regions. This helps researchers quickly interpret analysis results, optimize antibody sequence design, reduce risks in biopharmaceutical development, and accelerate the R&D process.

2 Parameters

• Nanobody: Specify whether the sequence is a nanobody. The default is “No.” If set to “Yes,” the tool will analyze the risk regions related to therapeutic nanobodies in the sequence.
• Hotspot: The tool can analyze the following potential PTM risk sites in the sequence:
  • Glycosylation
    Glycosylation sites, primarily recognizing the NXS/T motif (a common site for N-glycosylation), to detect glycosylation risk regions that may affect antibody stability and immunogenicity.
  • Deamidation
    Deamidation sites, including NG (asparagine deamidation) and NS motifs, to assess the potential for decreased stability or altered activity due to deamidation in the antibody sequence.
  • Isomerization
    Isomerization sites, covering DG (aspartate isomerization to asparagine) and DHK (aspartate isomerization to isoaspartate) motifs, to identify isomerization risks that may lead to structural changes in the antibody.
  • Oxidation
    Oxidation sites, including Met (methionine oxidation), His (histidine oxidation), and Trp (tryptophan oxidation), to detect key sites where oxidation may impact antibody activity or stability.
  • Cleavage
    Cleavage sites, such as DP (possibly a dipeptidyl peptidase cleavage site), to identify regions that may be susceptible to protease cleavage and assess stability risks in the antibody sequence.
  • Hydrolysis
    Hydrolysis sites, including TS and NP motifs, to detect regions where hydrolysis may lead to sequence cleavage or structural changes.
  • Cysteine
    Cysteine sites, specifically cysteine residues (C), related to disulfide bond formation or other redox reactions, to evaluate modifications that may affect the structural integrity of the antibody.
  • Cell Adhesion
    Cell adhesion-related sites, such as RGD (arginine-glycine-aspartic acid, a cell adhesion motif), to identify regions that may impact antibody-cell interactions.
• High-risk sites include: NG, DD, DG, DHK, NXS, NXT, Cys.

3 Results Explanation

Metric Explanation:
The Sequence Risk Site Ratio (Rate) is calculated to quantify the relative frequency of risk sites within a specific sequence. The formula for this metric is:

Rate = N/L

where N represents the number of risk sites, and L represents the length of the sequence.

Furthermore, to determine the safe area of the reference region for therapeutic antibody PTM (Post-Translational Modification) risk, the percentile method from statistics is employed. This is done by calculating the 97.5th percentile of the distribution of therapeutic antibody risk site ratios: Upper 95% = Percentile(X, 97.5), where X represents the sample set of therapeutic antibody risk site ratios. This reference value means that within the distribution of therapeutic antibody risk site ratios, 95% of the samples are below or equal to this value, while 5% of the samples are above it.

Sequence PTM Site Visualization:

Figure 1. PTM Hotspot Visualization of Example Sequence PTM Sites

Figure Caption:

• White Background: Indicates that there are no corresponding risk sites at that position.
• Legend in the Upper Left Corner: Provides color coding for different risk sites. Please refer to the legend to identify the colors corresponding to various risk sites.
• Low-Risk Sites: If the background color of a site matches a color in the legend, it indicates a low-risk site.
• High-Risk Sites: If there is a small circle above a site, it indicates a high-risk site.
• Inconsistent Colors: If the background color of a site is different from the color of the underline, it means that there are multiple risk sites at that position, each represented by a different color.

Visualization of the Reference Region for Therapeutic Antibody PTM Risk:
Note: The green shaded area in the lower left corner of the diagram represents the relatively safe area of the reference region for therapeutic antibody PTM risk.

Figure 2. PTM Hotspot Scatter Plot of the Risk Reference Area for Therapeutic Antibody Samples

Figure 3. PTM Hotspot Scatter Plot of the Risk Reference Area for CDRs of Therapeutic Antibody Samples