SpotLight Proteomics—A IgG-Enrichment Phenotype Profiling Approach with Clinical Implications

Abstract

Sarcoidosis is a systemic interstitial lung disease of unknown aetiology. Less invasive diagnostics are needed to decipher disease pathology and to distinguish sub-phenotypes. Here we test if SpotLight proteomics, which combines de novo MS/MS sequencing of enriched IgG and co-extracted proteins with subsequent label-free quantification of new and known peptides, can differentiate controls and sarcoidosis phenotypes (Löfgrens and non-Löfgrens syndrome, LS and nonLS). Intra-individually matched IgG enriched from serum and bronchial lavage fluid (BALF) from controls (n = 12), LS (n = 11) and nonLS (n = 12) were investigated. High-resolution mass-spectrometry SpotLight proteomics and uni- and multivariate-statistical analyses were used for data processing. Major differences were particularly observed in control-BALF versus sarcoidosis-BALF. However, interestingly, information obtained from BALF profiles was still present (but less prominent) in matched serum profiles. By using information from orthogonal partial least squares discriminant analysis (OPLS-DA) differentiating 1) sarcoidosis-BALF and control-BALF and 2) LS-BALF vs. nonLS-BALF, control-serum and sarcoidosis-serum (p = 0.0007) as well as LS-serum and nonLS-serum (p = 0.006) could be distinguished. Noteworthy, many factors prominent in identifying controls and patients were those associated with Fc-regulation, but also features from the IgG-Fab region and novel peptide variants. Differences between phenotypes were mostly IgG-specificity related. The results support the analytical utility of SpotLight proteomics which prospectively have potential to differentiate closely related phenotypes from a simple blood test.

Peptides, Antibodies, Peptide Antibodies and More

“…Upon prolonged immunization, specific clones may become dominant, which increases the specificity of the Abs, and immuno-affinity purification may be used to obtain monospecific PAbs [90]. In the case of a natural infection, the spectrum of PAbs can be characterised by proteomic methods, e.g., after isolation of IgG, as illustrated by Lundström et al in this special issue (SpotLight proteomics) [91] and used for production of recombinant Abs. Table 3 illustrates therapeutic PAb products obtained from human sera or by immunization of animals.…”

SpotLight proteomics identifies variable sequences of blood antibodies specific against deamidated human serum albumin

“…This work has already started, and information has been gathered on e.g. Alzheimer's disease [11] and sarcoidosis [12] using the so-called SpotLight proteomics approach.…”

“…Thus, obtained novel sequences that are common for most samples and strongly enriched in the patients compared to healthy controls are flagged as potential "hits". These hits are further in detail to remove possible false discoveries and compared with similarly obtained sequences from other diseases or infections [11,12]. The validated sequences are then introduced into the database of antibody sequences.…”

Template-Based Assembly of Proteomic Short Reads For De Novo Antibody Sequencing and Repertoire Profiling

“…Recently, we also demonstrated complete sequencing of a monoclonal antibody isolated from patient serum by reversed-phase LC fractionation and integrated bottom-up and top-down analysis . Plasma proteomics methods to profile polyclonal IgG mixtures and other heterogeneous variant proteins based on de novo methods (SpotLight and LAX) have also recently been described. − …”