Reliable Identification of Significant Differences in Differential Hydrogen Exchange-Mass Spectrometry Measurements Using a Hybrid Significance Testing Approach

Abstract

Differential hydrogen exchange-mass spectrometry (HX-MS) measurements are valuable for identification of differences in the higher order structures of proteins. Typically, the data sets are large with many differential HX values corresponding to many peptides monitored at several labeling times. To eliminate subjectivity and reliably identify significant differences in HX-MS measurements, a statistical analysis approach is needed. In this work, we performed null HX-MS measurements (i.e., no meaningful differences) on maltose binding protein and infliximab, a monoclonal antibody, to evaluate the reliability of different statistical analysis approaches. Null measurements are useful for directly evaluating the risk (i.e., falsely classifying a difference as significant) and power (i.e., failing to classify a true difference as significant) associated with different statistical analysis approaches. With null measurements, we identified weaknesses in the approaches commonly used. Individual tests of significance were prone to false positives due to the problem of multiple comparisons. Incorporation of Bonferroni correction led to unacceptably large limits of detection, severely decreasing the power. Analysis methods using a globally estimated significance limit also led to an overestimation of the limit of detection, leading to a loss of power. Here, we demonstrate a hybrid statistical analysis, based on volcano plots, that combines individual significance testing with an estimated global significance limit, that simultaneously decreased the risk of false positives and retained superior power. Furthermore, we highlight the utility of null HX-MS measurements to explicitly evaluate the criteria used to classify a difference in HX as significant.

FACT caught in the act of manipulating the nucleosome

“…5 ). Based on volcano plot analysis 16 , a significant decrease in deuterium uptake (ΔHX>0.3 Da and p-value <0.01) occurs in regions of the DDs and MDs of SPT16 and SSRP1. These primarily map to the inner face of the fork that interfaces directly with DNA and histones ( Fig.…”

Relation of Colloidal and Conformational Stabilities to Aggregate Formation in a Monoclonal Antibody

“…We used the volcano plot which combined a probability value (p-value) determined from Welch's t-test and a scatter-plot of DHX for statistical analysis. 35 Significance levels were defined at a ¼ 0.01 for horizontal p-value and at ±0.289 Da for vertical DHX value as shown in the Supplementary Information.…”

Structural dynamics of cytochrome P450 3A4 in the presence of substrates and cytochrome P450 reductase

“…This hybrid statistical test combines individual significance testing with a globally estimated threshold calculated from the variance of all data points. 40 All significant peptides detected in our study are detailed in Figures S3 and S4 , and comparisons between results obtained under different conditions can be visualized in butterfly plots ( Figure S5 ). The significant uptake changes over time were summed in order to capture more subtle changes in HDX.…”