ßB1-Crystallin Quantification in Human Lens by 18O Labeling and LC-MS

Abstract

Purpose:: New methods are required to quantify crystallins in human lens to track changes in composition, abundance and solubility during aging and cataract. Measurement of absolute crystallin abundance was tested using stable isotope labeled internal peptide standards to quantify ßB1-crystallin.

Methods:: Tryptic peptides ßB1- ¹⁵⁰ISLFEGANFK¹⁵⁹ and ²⁰²GYQYLLEPGDFR²¹³, were synthesized, purified by reverse phase chromatography, dried, and the two C-terminal ¹⁶O atoms exchanged with ¹⁸O by incubation in H₂¹⁸O water with immobilized trypsin. An amino acid analysis was performed to determine the concentrations of the labeled peptides. Soluble protein from a 3-day old human lens was digested to completion with trypsin. Based on a BCA protein assay, 2 µg of the lens digest was spiked with 10 pmol of each synthetic ßB1 peptide. The spiked lens digest was then separated by capillary reverse phase chromatography and MS data collected using an LTQ ion trap (ThermoFinnigan), by alternating full-range zoom scans and data-dependent tandem MS scans. The averaged spectra for the light and heavy forms of the peptides were used to calculate the abundance of endogenous ßB1 peptides in the digest.

Results:: Peptides labeled with ¹⁸O co-elute with their corresponding unlabeled peptides, and due to the small +4 Da mass shift, result in partially overlapping isotopic distributions. The theoretical isotopic distributions for each ßB1 peptide (unlabeled and ¹⁸O labeled forms) were calculated, and the overlapping isotopic distributions deconvoluted using least squares fitting to determine the ion current associated with each peptide form. The ion current ratios of labeled peptide (known standard) to unlabeled peptide (3-day old lens) for 150-159 and 202-213 peptides were 0.617 and 0.756, respectively. Therefore the amount of ßB1 in the 2 µg lens sample from a 3-day old lens was calculated as 14.6 pmol, corresponding to an abundance of 20.5% ßB1-crystallin in young human lens.

Conclusions:: Inexpensive ¹⁸O labeling of synthetic ßB1-crystallin peptides enabled absolute quantification of ßB1 from a complex human lens digest. The estimation that 1/5 of human lens was comprised of ßB1-crystallin was unexpected, given that previous gel-based quantification have estimated an approximate 8% abundance. Additional measurements using a greater number of synthetic peptides and accurate evaluation of lens protein content by amino acid analysis will be used to validate ¹⁸O labeling of synthetic peptides as a method to track changes in crystallin abundance during aging and cataract.

Keywords: crystallins • proteomics