Discovering Mercury Protein Modifications in Whole Proteomes Using Natural Isotope Distributions Observed in Liquid Chromatography-Tandem Mass Spectrometry

Abstract

The identification of peptides that result from post-translational modifications is critical for understanding normal pathways of cellular regulation as well as identifying damage from, or exposures to xenobiotics, i.e. the exposome. However, because of their low abundance in proteomes, effective detection of modified peptides by mass spectrometry (MS) typically requires enrichment to eliminate false identifications. We present a new method for confidently identifying peptides with mercury (Hg)-containing adducts that is based on the influence of mercury's seven stable isotopes on peptide isotope distributions detected by high-resolution MS. Using a pure protein and E. coli cultures exposed to phenyl mercuric acetate, we show the pattern of peak heights in isotope distributions from primary MS single scans efficiently identified Hg adducts in data from chromatographic separation coupled with tandem mass spectrometry with sensitivity and specificity greater than 90%. Isotope distributions are independent of peptide identifications based on peptide fragmentation (e.g. by SEQUEST), so both methods can be combined to eliminate false positives. Summing peptide isotope distributions across multiple scans improved specificity to 99.4% and sensitivity above 95%, affording identification of an unexpected Hg modification. We also illustrate the theoretical applicability of the method for detection of several less common elements including the essential element, selenium, as selenocysteine in peptides. Molecular & Cellular Proteomics 10: 10.1074/mcp.M110.004853, 1-13, 2011.The availability of entire genomic sequences and the development of tandem mass spectrometry and liquid chromatography (LC-MS/MS) 1 , has enabled the widespread use of shotgun or bottom-up proteomics over the last decade (1). Despite imperfect peptide identifications, these techniques effectively catalogue even rare proteins present in cells because most proteins are represented by several unique peptides, diminishing the effects of any single false peptide identification. Recent efforts have also enhanced shotgun proteomics to resolve fine scale protein features, such as post-translational modifications (PTM) (2). However, because many modifications often appear in only a single peptide in typical shotgun proteomics preparations, false peptide identifications can greatly impact the characterization of these modifications.Preprocessing proteomic preparations to enrich peptides with a PTM of interest is one common strategy used to minimize false identifications. If the modification cannot be enriched, then other independent evidence is needed to increase confidence in PTM identifications. Types of data that have been used for PTM corroboration include characteristic fragmentation such as that for the neutral loss of phosphate (3), or modifications observed on overlapping peptides resulting from digestions with different proteases (4). Here we present a method to discern the anomalous isotope distribution of peptides either post-translationally or cotransl...

Organic and inorganic mercurials have distinct effects on cellular thiols, metal homeostasis, and Fe-binding proteins in Escherichia coli

“…Cultures were prepared as above for 15 min exposure to 40 µM PMA, 160 µM merthiolate or 20 µM mercuric acetate, but harvested cells were suspended in ammonium bicarbonate buffer amended with iodoacetamide (IAM) to prevent redistribution of mercury adducts by exchange with free thiols during preparation for liquid chromatography-coupled mass spectrometry (LC-MS/MS) proteomic analysis [28] (Zink et. al., manuscript in preparation).…”

“…Despite many studies on individual enzymes and cellular processes, there has been no comprehensive study of the bulk effects of Hg on the thiol pool and metal homeostasis in any organism. As part of a larger proteomics project to define the mercury exposome [28], we examined the effects of brief, acute exposure of growing cells to inorganic and organomercurials (Fig. S1) on the cellular content of thiols, of essential metals, and of free iron and iron-binding proteins in E. coli K-12 MG1655.…”

Infectome: A platform to trace infectious triggers of autoimmunity

“…The exposome provides a potential platform by which these exposures may be measured. It has been suggested that their by-products in an individual, such as DNA and protein modification, can serve as biomarkers that are potentially measurable [38] in the blood or body fluids of patients [3,[36][37][38] via technologies such as liquid chromatography-tandem mass spectrometry (LC-MS/MS) [42], DNA adducts [43], functional measurements of antioxidant capacity, and breath analysis [34].…”

Tracing environmental markers of autoimmunity: introducing the infectome

“…Components of the exposome may also serve as measurable biomarkers [33] in the blood or body fluids of affected Etio Pathogenesis of Autoimmunity (2013) 56: 220-240 221 patients or individuals prone to develop autoimmune diseases [4,[31][32][33]. Technologies such as liquid chromatography-tandem mass spectrometry [37], DNA adducts [38], functional measurements of antioxidant capacity and breath analysis may be utilised in such instances [29]. Two methods of exposomal measurement have been proposed: the ''bottom-up'' method which measures the external factors [30] and the ''top-down'' method which measures internal factors.…”