Dataset Information

Rapid Tear Proteomics via In-Capillary Digestion for Biomarker Discovery

ABSTRACT: Tear fluid is emerging as a valuable resource for biomarker discovery, but its low sample volume and dynamic composition pose significant challenges for analytical testing. Most tear proteomics studies have focused on samples collected with Schirmer strips, while capillary tube collection has received less attention. To address these challenges, we developed a novel in-capillary trypsin digestion workflow that requires as little as 0.5 μL of tear fluid for bottom-up shotgun proteomics. This method uses liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify a higher number of proteins with enhanced efficiency compared to previously reported methods, which typically required 5-10 μL of pooled tear fluid. Using the same microcentrifuge tube for both tear collection and sample processing, this streamlined workflow simplifies sample handling and minimizes both sample loss and experimental errors associated with sample transfer. This method also efficiently reduced sample processing time to under 2 hours prior to overnight trypsin di-gestion, compared to the 5-8 hours required by other methods. With this workflow, we identified 500-800 proteins per 0.5 μL sample without the need for fractionation, allowing for at least three technical replicates. This optimized workflow greatly enhances the efficiency of tear proteomics for biomarker discovery.

INSTRUMENT(S): ultraflex, maXis, LTQ, LCQ Classic, Orbitrap Fusion, 6520A Quadrupole Time-of-Flight LC/MS, Orbitrap Fusion Lumos, Synapt MS, timsTOF Pro 2, 6410 Triple Quadrupole LC/MS, LTQ Orbitrap Elite, Q Exactive, 6340 Ion Trap LC/MS, LTQ Orbitrap, autoflex, LTQ Orbitrap Velos, 4800 Proteomics Analyzer, Q-Tof Ultima, LTQ FT, 6220 Time-of-Flight LC/MS, TripleTOF 5600, Q Exactive HF, MALDI Synapt MS, Q TRAP, QSTAR, 4700 Proteomics Analyzer

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Tear, Tear Secreting Cell

SUBMITTER: James Xiao

LAB HEAD: Leanne T. Labriola

PROVIDER: PXD056978 | Pride | 2025-05-07

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	AG-1000ng-21min-R1-DDA.d.zip	Other
	AG-1000ng-21min-R2-DDA.d.zip	Other
	AG-1000ng-21min-R3-DDA.d.zip	Other
	AG-1000ng-44min-R1-DDA.d.zip	Other
	AG-1000ng-44min-R2-DDA.d.zip	Other

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Publications

High-Throughput Tear Proteomics via In-Capillary Digestion for Biomarker Discovery.

Xiao James J Frenia Kyla K Garwood Kathleen C KC Kimmel Jeremy J Labriola Leanne T LT

International journal of molecular sciences 20241114 22

Tear fluid has emerged as a valuable resource for biomarker discovery; however, the limited sample volume, the dynamic composition, and the variability introduced by collection methods all present significant challenges to the analysis and interpretation of the results. A majority of tear proteomic studies have utilized Schirmer strips for tear fluid collection; however, microcapillary collection can provide a superior collection method for proteomic studies when analysis procedures are optimize ...[more]

PMID: 39596304

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Project description:Glycosylation of proteins is one of the most common post-translational modification (PTM) and plays important regulatory functions in diverse biological processes such as protein stability or cell signaling. Accordingly, glycoproteins are also a consistent part of the human tear film proteome maintaining the proper function of the ocular surface and forming the first defense barrier of the ocular immune system. Irregularities in the glycoproteomic composition of the tear film might promote development of chronic eye diseases indicating glycoproteins as valuable source for biomarker discovery or drug target identification. The present study aimed to develop a lectin-based affinity method for the enrichment and concentration of tear glycoproteins/glycopeptides and the characterization of their specific N-glycosylation sites by high-resolution mass spectrometry (MS). For method development and evaluation, we accumulated first native glycoproteins from human tear sample pools and assessed the enrichment efficiency of different lectin column systems by 1D gel electrophoresis and specific protein stainings (Coomassie and glycoproteins). The best-performing multi-lectin column system (comprising the four lectins ConA, JAC, WGA and UEA I, termed as 4L) was applied for glycopeptide enrichment from human tear sample digests followed by MS-based detection and localization of their specific N-glycosylation sites. As main result, the present study identified in total 26 N glycosylation sites of 11 N-glycoproteins in tear sample pools of healthy individuals (n=3 biological sample pools). Amongst others, we identified tear film proteins lactotransferrin (N497 and N642, LTF), Ig heavy chain constant α-1 (N144 and 340, IGHA1), prolactin-inducible protein (N105, PIP) as well as extracellular lacritin (N105, LACRT) as highly reliable and significant N glycoproteins, which were already associated with the pathogenesis of various chronic eye diseases such as the dry eye syndrome (DES). In conclusion, the results of the present study will serve as important tear film N-glycoprotein catalogue for future studies focusing the human tear film and ocular surface-related inflammatory diseases.