Refinement of a Methodology for Untargeted Detection of Serum Albumin Adducts in Human Populations

Abstract

Covalently modified blood proteins (e.g., serum albumin adducts) are increasingly being viewed as potential biomarkers via which the environmental causes of human diseases may be understood. The notion that some (perhaps many) modifications have yet to be discovered has led to the development of untargeted adductomics methods, which attempt to capture entire populations of adducts. One such method is fixed-step selected reaction monitoring (FS-SRM), which analyses distributions of serum albumin adducts via shifts in the mass of a tryptic peptide [Li et al. (2011) Mol. Cell. Proteomics 10, M110.004606]. Working on the basis that FS-SRM might be able to detect biological variation due to environmental factors, we aimed to scale the methodology for use in an epidemiological setting. Development of sample preparation methods led to a batch workflow with increased throughput and provision for quality control. Challenges posed by technical and biological variation were addressed in the processing and interpretation of the data. A pilot study of 20 smokers and 20 never-smokers provided evidence of an effect of smoking on levels of putative serum albumin adducts. Differences between smokers and never-smokers were most apparent in putative adducts with net gains in mass between 105 and 114 Da (relative to unmodified albumin). The findings suggest that our implementation of FS-SRM could be useful for studying other environmental factors with relevance to human health.

EXPOsOMICS: final policy workshop and stakeholder consultation

“…Adductomics, the untargeted detection of DNA or protein adducts of endogenous or exogenous origin, is a new field in exposome research. Many of the studies in this area focus on the untargeted analysis of protein adducts in human serum albumin (Cys34 - the major site of modification) through a method developed in 2011 [ 23 ] that has been adapted for higher-throughput use in EXPOsOMICS adductomics studies [ 24 ]. The half-life of albumin (20–25 days) means that measurements of its covalent adducts reflect a longer “capture period” of exposure than other more transient omics biomarkers.…”

Protein Adductomics: Analytical Developments and Applications in Human Biomonitoring

“…For this reason, it is common to see lists of equally-spaced integer or half-integer m / z values [28]. We have referred to these arbitrary values as sampling points [33]. The idea of an arbitrary SRM experiment might strike the reader as odd, since SRM is traditionally used for targeted analyses, but for untargeted analyses it does not matter where the sampling points fall.…”

“…Our personal experience with protein adductomics has been in the implementation of FS-SRM for epidemiological studies [10,33]. Such studies, which typically involve tens or hundreds of samples, pose challenges that are not necessarily encountered in smaller pilot studies.…”

“…Funk et al [70] sought to limit this effect by removing the S-thiolation prior to the scavenging step. In our hands, the thiol scavenging method proved difficult to implement in a high-throughput setting, and so we deleted it from our workflow [33]. Chung et al [71] used thiol scavenging as the first of two stages of enrichment, the second stage being an antibody-mediated purification of the substituted T3 peptides using a polyclonal antibody raised against the T3 peptide but having cross-reactivity with adducts.…”

“…Accuracy, in practice, may suffer as a consequence of the need to capture a range of adducts. It is likely that the use of generic standards (e.g., the S-carbamidomethylated T3 peptide for FS-SRM) affects accuracy, and therefore precludes absolute quantification [33]. Dynamic ranges are dependent on the analytical method, and presumably also on the ability to enrich adducts.…”