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This abstract is assigned to session AOB and FRO E-Posters

Title	Top-down proteomics of human tears in a clinical context.
Abstract Nr.	3038

Purpose	To analyze the different (iso)forms of the proteins in clinically sampled tears. In several pathologies little effect is seen at the level of the gene, transcript or even protein identities that can be detected. Often, however, disease-specific protein modifications do result in modified ‘proteoforms’, with altered bioactivity.
Methods	Tears of multiple donors were analyzed with bottom-up, as well as top-down proteomics. As such >10 different biological samples were analyzed in duplicate by reversed phase capillary LC / quadrupole-orbitrap MS.
Results	The overall bottom-up analysis yielded > 350 different protein identities, as previously reported. Top-down data enabled 90 proteins to be confidently identified, with a bias towards the higher abundant protein species. Surprisingly these 90 proteins appear in >750 distinct proteoforms. Some proteoforms have nearly full top-down MSMS sequence coverage; others lack coverage in certain protein domains, not seldom rich in cysteines (S-S bridges). Only very few proteins occur in a single proteoform. One of the proteins appearing in multitude of isoforms is lacritin, an important protein in Dry Eye Disease because of its autocrine effect on tear secretion. Indeed, >175 different lacritin proteoforms were detected (all donors cumulated). Several proteoforms are unique for a specific donor, probably representing inter-individual genomic/genetic variations.
Conclusion	A large amount of novel biological information remains hidden when classical (bottom-up) proteomics is done, and part of it may get unvailed by a top-down approach.
Conflict of interest	No

Authors 1

Last name	RAUS
Initials	PPM
Department	Miró Centrum voor Oogheelkunde en Ooglidchirurgie
City	Geel, België

Authors 2

Last name	de Winde
Initials	JH
Department	Univ. Leiden
City	Leiden, Nederland

Authors 3

Last name	Verhaert
Initials	PDEM
Department	ProteoformiX
City	Beerse, Belgie