Measurement of absolute abundance of crystallins in human and αA N101D transgenic mouse lenses using ¹⁵N-labeled crystallin standards.

ABSTRACT: Stable isotope labeled standards of all major human lens crystallins were created to measure the abundance of lens endogenous crystallins from birth to adulthood. All major human crystallins (αA, αB, βA2, βA3/A1, βA4, βB1, βB2, βB3, γA, γB, γC, γD, γS) were cloned with N-terminal 6 x His tagged SUMO for ease of purification and the ability to generate natural N-termini by SUMO protease cleavage when producing crystallins for structure/function studies. They were then expressed in ¹⁵N-enriched media, quantified by mass spectrometry, and mixed in proportions found in young human lens to act as an artificial lens standard. The absolute quantification method was tested using soluble protein from 5-day, 23-day, 18-month, and 18-year-old human lenses spiked with the ¹⁵N art

SUBMITTER: Halverson-Kolkind KA 

PROVIDER: S-EPMC11724759 | biostudies-literature | 2024 Nov

REPOSITORIES: biostudies-literature