Introducing the concept of centergram: a new tool to squeeze data from separation techniques-mass spectrometry couplings

Abstract

tIn separation techniques hyphenated to mass spectrometry (MS) the bulk from the separation step iscontinuously flowing into the mass spectrometer where the compounds, arriving at each separationtime, are ionized and further separated based on their m/z ratio. An MS detector is recognized as beinga universal detector, although it can also be a very selective instrument. In spite of these advantages,classical two dimensional representations from these hyphenated systems, such as those based on thebase peak of electropherogram/chromatogram or on the total ion of electropherogram/chromatogram,usually hide a large number of features that if correctly assessed will show the presence of co-migratingspecies and/or the low abundant ones. The uses of peak picking algorithms to detect and measure as manypeaks as possible from a dataset allow extracting much more information. However, a single migratingcompound usually produces a multiplicity of ions, making difficult to differentiate peaks generated bythe same compound from other peaks due e.g., to closely co-migrating/eluting species. In this work, anew representation is proposed and its usefulness demonstrated with experimental data from capil-lary electrophoresis-hyphenated to a time of flight mass spectrometer via an electrospray interface. Thisrepresentation, called centergram, is obtained after using a peak picking methodology that detects elec-trophoretic peaks of single ions and measure their positions. The centergram is the histogram (i.e. thecount of the number of observations that fall into each one of the intervals, known as bins, as deter-mined by the user) of the measured positions. The intensity of the bars in this histogram will indicatethe amount of peaks in the whole dataset whose centers are within each interval. As a compound thathas been separated and has entered the MS instrument will produce multiple images at the same posi-tion along the m/z dimension, the centergram will exhibit a series of intense bars around the migrationtime. Those bars will allow defining a centergram peak whose area will be proportional to the numberof different types of ions that have been generated in the ionization chamber, the position will be equalto the migration/retention time of the parent compounds and the width will depend on the precisionin the measurement of the peak positions. The efficiency of this peak is determined to be up to thirtytimes higher than the equivalent peak in the classical base peak electropherogram allowing detectingeasily co-migrating peaks or the presence of compounds at very low abundance. The number of peaksdetected by using this new tool called centergram was increased by more than a factor of 3 compared tothe standard representations.