PXD053890

PXD053890 is an original dataset announced via ProteomeXchange.

Title	Absolute abundance of human crystallins in lens
Description	Purpose: Measurement of crystallin abundances in human lens has previous relied on poorly reproducible electrophoretic separations or only semi-quantitative mass spectrometry. Therefore, isotope labeled standards were created for all major human lens crystallins so that their absolute abundances could be accurately measured in lens from birth to adulthood. Methods: 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. They were then expressed in 15N-enriched media, quantified by mass spectrometry, and mixed in proportions found in young human lens to act as a standard. Soluble protein from 5-day, 23-day, 18-month, and 18-year-old human lenses was then spiked with these mixed 15N labeled crystallin standards, proteins trypsinized, relative ratios of light and heavy labeled peptides determined using high-resolution precursor and data-independent MS2 scans and data analysis using Skyline software. Human and mouse αA was also measured in the transgenic mouse αA N101D cataract model. Results: Crystallin abundances were accurately measured with average coefficients of variation of approximately 2% across all 13 crystallins. αA crystallin comprised 27% of the soluble protein of 5-day-old lens and decreased to 16% by 18-years of age. Over this time period αB increased from 6% to 9% and the αA/αB ratio decreased from 5/1 to 2/1. γS-crystallin also increased 2-nearly 2-fold from 7% to 12%, becoming the 3rd most abundant protein in adult lens, while βB1 increased from 14% to 20%, becoming the most abundant crystallin of adult lens. Minor crystallins βA2, βB3, and γA comprised only about 1% each of the newborn lens soluble protein, and their abundance dropped precipitously by adulthood. While 9 of the SUMO tagged crystallins were useful for purification of crystallins for structural studies, γA, γB, γC, and γD were resistant to cleavage by SUMO protease. The abundance of WT and N101D human αA in transgenic mouse lenses was approximately 40-fold lower than endogenous mouse αA, but the deamidation mimic human αA N101D was less soluble than human WT αA. Conclusions: The high content of αA and the transient abundance of βA2, βB3, and γA in young lens suggest these crystallins play a role in early lens development and growth. βB1 becoming the most abundant crystallin may result from its role in promoting higher order β-crystallin oligomerization in mature lens. The full set of human crystallin expression vectors in the Addgene repository should be a useful resource for crystallin purification and study. The 15N labeled crystallin standards will allow accurate quantitation of crystallins in lens anatomic regions, as well as measuring the composition of insoluble light scattering crystallin aggregates in cataractous lenses. They can also be used to measure the abundance of crystallins expressed in transgenic animal models.
HostingRepository	PanoramaPublic
AnnounceDate	2024-10-08
AnnouncementXML	Submission_2024-10-08_01:00:21.373.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Supported dataset by repository
PrimarySubmitter	Larry David
SpeciesList	scientific name: Mus musculus; NCBI TaxID: 10090; scientific name: Homo sapiens; NCBI TaxID: 9606;
ModificationList	Carbamidomethyl; Label:15N(1); Label:15N(2); Label:15N(3); Label:15N(4)
Instrument	Orbitrap Eclipse

Revision	Datetime	Status	ChangeLog Entry
0	2024-07-11 11:02:07	ID requested
⏵ 1	2024-10-08 01:00:22	announced

Halverson-Kolkind KA, Caputo N, Lampi KJ, Srivastava O, David LL, N-labeled crystallin standards. Exp Eye Res, 248():110115(2024) [pubmed]

submitter keyword: Crystallin, lens, cataract, stable isotope labeling, mass spectrometry

Larry David
contact affiliation	Oregon Health & Science University
contact email	davidl@ohsu.edu
lab head
Larry David
contact affiliation	Oregon Health & Science University
contact email	davidl@ohsu.edu
dataset submitter

Panorama Public dataset URI