Suppression correction and characteristic study in liquid chromatography/Fourier transform mass spectrometry measurements

Over the past decade or so, liquid chromatography/Fourier transform mass spectrometry (LC/FTMS) has gained increasing popularity for its high mass resolution and mass accuracy which allow for sensitive peptide identification both in LC/MS and tandem mass spectrometry (MS/MS) experiments. As the relationship between actual peptide abundance and the corresponding spectrum intensity is approximately linear,[1] FTMS is also widely used in quantitative proteomics and biomarker discovery. However, many researchers have found that FTMS lacks accuracy in ion abundance measurements, which is essential in peptide quantification and LC/MS peptide identification. Padley et al.[2] made note of several sources of nonlinearity in measurements. Schrader et al.[3] also mentioned signal loss in a large compound library experiment. Sterner et al.[4] found that the signals of small proteins are suppressed by larger ones. Gordon et al. reported that due to ion interaction, the spectral signal intensities do not necessarily reflect true trapped-ion abundances.[1,5] Additional signal suppression phenomena due to the effects of measuring several peptides are reported in Schmid et al.[6] In Bresson et al.,[7] the suppression is explained by ion cloud stability: “… Clouds that contain the greater number of ions will tend to have higher charge densities and stability, compared with less populated ion clouds. Smaller ion clouds are more susceptible to break up because of shear forces, including those arising from adjacent clouds. The magnitude of signal decay effects is therefore anticipated to be greatest for clouds containing the smallest number of ions, corresponding to weaker signals than predicted from a theoretical distribution.” The problem is addressed by using a shorter time domain signal before Fourier transform in Bresson et al.[7] However, nowadays, such a signal before Fourier transform is generally not available and using a shorter time signal still cannot eliminate the suppression completely. Our investigation of LC/FTMS datasets (collected on an Orbitrap) reveals that the suppression of low abundance peaks acts to distort the abundance ratios between isotopes. The distortion on isotope ratios will severely limit the reliability of FTMS quantification results in labeled experiments such as SILAC or O18. It is critical to study the nature of the suppression and its effect on abundance measurements. In this paper, we investigate: (1) if the suppression function is homogenous for different peptides; (2) if the suppression is homogenous for different isotopes of the same peptide; (3) a possible correction strategy of such distortions; and (4) the suppression characteristics of FTMS instruments. We find that the suppression is non-homogenous for different peptides, but it is homogenous for isotopes of the same peptide. Also, we develop an iterative algorithm that can correct the suppression for some peptides with relative high abundance. Since the suppression causes distortion on measured isotope ratios, this greatly affects the accuracies of differential analysis based on FTMS. We investigate the mean and variance of the abundance distortions at different intensities, which is critical for the correct interpretation of FTMS quantification results in differential analysis. Although the datasets we have investigated were all collected on Orbitrap, abundance suppression of lower intensity ions has also been documented in Fourier transform ion cyclotron resonance (FT-ICR)-MS.[5,7] Thus, the developed suppression correction and characterization approach should be applicable to FTMS in general. The datasets, manuscript and supporting materials which contain more detailed plots of this paper are available from the internet.[8]