A novel approach for the chromatographic purification and peptide mass fingerprinting of urinary free light chains

• Published in: International journal of biological macromolecules 2017-02, Vol.95, p.331-339
• Main Authors: Mali, Bhupesh C., Badgujar, Shamkant B., Shukla, Kunal K., Bhanushali, Paresh B.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Bence Jones proteins; Chromatography - methods; Free light chains; Humans; Immunoblotting; Immunoglobulin kappa-Chains - chemistry; Immunoglobulin kappa-Chains - isolation & purification; Immunoglobulin kappa-Chains - urine; Immunoglobulin lambda-Chains - chemistry; Immunoglobulin lambda-Chains - isolation & purification; Immunoglobulin lambda-Chains - urine; Medical Waste; Peptide Mapping - methods; Proteomics; Urine
• ISSN: 0141-8130; 1879-0003
• DOI: 10.1016/j.ijbiomac.2016.11.076

[Display omitted] •Urinary free light chains (λ-FLCs and κ-FLCs) have been purified.•Detection of urinary FLCs was achieved by immunoblotting using FLC specific monoclonal antibody.•Peptide mass fingerprint analysis of λ-FLCs and κ-FLCs has yielded peptides that partially match with gi|218783338 and gi|48475432 respectively.•This is the first record on characterization and validation of Urinary FLCs. We describe a chromatographic approach for the purification of urinary free light chains (FLCs) viz., lambda free light chains (λ-FLCs) and kappa free light chains (κ-FLCs). Isolated urinary FLCs were analyzed by SDS-PAGE, immunoblotting and mass spectrometry (MS). The relative molecular masses of λ-FLC and κ-FLC are 22,933.397 and 23,544.336Da respectively. Moreover, dimer forms of each FLC were also detected in mass spectrum which corresponds to 45,737.747 and 47,348.028Da respectively for λ-FLCs and κ-FLCs. Peptide mass fingerprint analysis of the purified λ-FLCs and κ-FLCs has yielded peptides that partially match with known light chain sequences viz., gi|218783338 and gi|48475432 respectively. The tryptic digestion profile of isolated FLCs infers the exclusive nature of them and they may be additive molecules in the dictionary of urinary proteins. This is the first report of characterization and validation of FLCs from large volume samples by peptide sequencing. This simple and cost-effective approach to purification of FLCs, together with the easy availability of urine samples make the large-scale production of FLCs possible, allowing exploration of various bioclinical as well as biodiagnostic applications.