Comprehensive analysis of LC/MS data using pseudocolor plots

Abstract

We have developed new applications of the pseudocolor plot for the analysis of LC/MS data. These applications include spectral averaging, analysis of variance, differential comparison of spectra, and qualitative filtering by compound class. These applications have been motivated by the need to better understand LC/MS data generated from analysis of human biofluids. The examples presented use data generated to profile steroid hormones in urine extracts from a Cushing's disease patient relative to a healthy control, but are general to any discovery-based scanning mass spectrometry technique. In addition to new visualization techniques, we introduce a new metric of variance: the relative maximum difference from the mean. We also introduce the concept of substructure-dependent analysis of steroid hormones using precursor ion scans. These new analytical techniques provide an alternative approach to traditional untargeted metabolomics workflow. We present an approach to discovery using MS that essentially eliminates alignment or preprocessing of spectra. Moreover, we demonstrate the concept that untargeted metabolomics can be achieved using low mass resolution instrumentation.

Figure 1

Pseudocolor plots of mass spectral profile intensities of extracted urine from a Cushing’s disease patient. The plots include the m/z values 340–375 and RT of 9–14 min. Plots are of (a) the intensity of a single replicate and (b) the arithmetic mean intensity of technical triplicates of the same extract

Figure 2

Quality control pseudocolor plots of mass spectral profile intensities from extracted urine of a Cushing’s disease patient analyzed in technical triplicate. The plots include the m/z values 340–375 and RT of 9–14 mi. (a) A plot of the coefficient of variation of the intensity values, (b) A RMDM plot (see text for a description of this quantity)

Figure 3

RMDM distributions of spectral profile intensities from a urine extract from a Cushing’s disease patient analyzed in technical triplicate. The points in black have been plotted with no flooring. The values in blue have been floored to 30 counts. The values in green have been floored to 60 counts. The lighter hues are those values unaffected by flooring. These distributions are displayed both as (a) a scatter plot where the y-axis is RMDM and the x-axis is an intensity-sorted index and (b) as histograms

Figure 4

A pseudocolor plot of the Log₂ transformed ratio of the arithmetic mean of intensities from mass spectral profiling of extracted urine from a Cushing’s disease patient and a healthy control. Each urine extract was analyzed in technical triplicate. The plot includes the m/z values 340–375 and RT of 9–14 min

Figure 5

Evaluation of the product ion intensity as it relates to steroidal molecular substructure. The values are base peak normalized intensities of product ion spectra at CE=40 eV of pure standards. Green values are those m/z values of the product ion spectra that are ≥10 % of the base peak of the product ion spectrum of an analyte. Note that only those analytes that have the A, B, and C ring substructure of Cortisol produce a significant product ion at m/z= 121.1 at this collision energy

Figure 6

A pseudocolor plot that shows substructure-dependent mass spectral profiling of extracted urine from a Cushing’s disease patient as it relates to a healthy control. The plot includes the m/z values 340–375 and RT of 9–14 min. The areas in black are regions where the technical triplicate arithmetic mean intensity of the Cushing’s disease urine extract is 4-fold greater than that of a healthy control and that the precursor ion scan intensity of the 4-ene-11-ol-3-one specific transition is greater than the 1,3,5(10)-triene-3ol or 4-ene-3-one specific transitions. Unknown-361-A was a mass spectral feature that did not exist in our initial set of standards and following further analysis was determined to be cortisone