sample-preparation-for-mass-spectrometry-based-proteomics-analysis

Sample Preparation for Mass-spectrometry-based Proteomics Analysis of Ocular Microvessels

The use of isolated ocular blood vessels in vitro to decipher the pathophysiological state of the eye using advanced technological approaches has greatly ...

University Medical Centre of the Johannes Gutenberg University Mainz

Short posterior ciliary arteries (sPCA) provide the major blood supply to the optic nerve head. Emerging evidence has linked structural and functional anomalies of sPCA to the pathogenesis of several ocular disorders that cause varying degrees of visual loss, particularly anterior ischaemic optic neuropathy and glaucoma. Although the functional relevance of this vascular bed is well-recognized, the proteome of sPCA remains uncharacterized. Since the porcine ocular system closely resembles that of the human's and is increasingly employed in translational ophthalmic research, this study characterized the proteome of porcine sPCA employing the mass spectrometry-based proteomics strategy. A total of 1742 proteins and 10527 peptides were identified in the porcine sPCA. The major biological processes involved in the maintenance of physiological functions of the sPCA included redox and metabolic processes, and cytoskeleton organization. These proteins were further clustered into diverse signalling pathways that regulate vasoactivity of sPCA, namely the tight junction, α- and β-adrenoceptor, 14-3-3, nitric oxide synthase and endothelin-1 -mediated signalling pathways. This study provides the first insight into the complex mechanisms dictating the vast protein repertoire in normal vascular physiology of the porcine sPCA. It is envisioned that our findings will serve as important benchmarks for future studies of sPCA.

First insight into the proteome landscape of the porcine short posterior ciliary arteries: Key signalling pathways maintaining physiologic functions.

Contact lenses (CLs) provide a superior alternative to spectacles. Although beneficial, the global burden of ocular dysfunctions attributed to regular use of CLs remains a topic of much challenge in ophthalmic research owing to debilitating clinical repercussions on the ocular surface, which are often manifested as breach in tear film integrity. This study elucidated the intricate tear proteome changes attributed to the use of different CLs (hard and soft) and unravelled, for the first time, the restorative mechanisms of several protein clusters following acute renouncement of CL use employing the label-free mass spectrometry-based quantitative proteomics approach. The expression patterns of certain proteins clusters were specific to the use of a particular lens type and a large majority of these actively regulates cell death and survival and, modulates cellular movement on the ocular surface. Noteworthy, CL use also evoked a significant upregulation of glycolytic enzymes associated with hypoxia and corresponding cognate metabolic pathways, particularly glucose metabolism and FXR/RXR pathways. Importantly, the assessment of CL renouncement unravelled the restorative properties of several clusters of proteins involved mainly in organismal injury and abnormalities and, cellular function and maintenance. These proteins play key roles in restoring tear homeostasis and wound-healing mechanisms post-CL use-elicited injury.

Proteomics Unravels the Regulatory Mechanisms in Human Tears Following Acute Renouncement of Contact Lens Use: A Comparison between Hard and Soft Lenses.

Proper sample preparation protocols represent a critical step for liquid chromatography-mass spectrometry (LC-MS)-based proteomic study designs and influence the speed, performance and automation of high-throughput data acquisition. The main objective of this study was to compare two commercial solid-phase extraction (SPE)-based sample preparation protocols (comprising SOLAµ HRP SPE spin plates from Thermo Fisher Scientific and ZIPTIP C18 pipette tips from Merck Millipore) for analytical performance, reproducibility, and analysis speed. The house swine represents a promising animal model for studying human eye diseases including glaucoma and provides excellent requirements for the qualitative and quantitative MS-based comparison in terms of ocular proteomics. In total six technical replicates of two protein fractions [extracted with 0.1% dodecyl-ß-maltoside (DDM) or 1% trifluoroacetic acid (TFA)] of porcine retinal tissues were subjected to in-gel trypsin digestion and purified with both SPE-based workflows ( = 3) prior to LC-MS analysis. On average, 550 ± 70 proteins (1512 ± 199 peptides) and 305 ± 48 proteins (806 ± 144 peptides) were identified from DDM and TFA protein fractions, respectively, after ZIPTIP C18 purification, and SOLAµ workflow resulted in the detection of 513 ± 55 proteins (1347 ± 180 peptides) and 300 ± 33 proteins (722 ± 87 peptides), respectively (FDR < 1%). Venn diagram analysis revealed an average overlap of 65 ± 2% (DDM fraction) and 69 ± 4% (TFA fraction) in protein identifications between both SPE-based methods. Quantitative analysis of 25 glaucoma-related protein markers also showed no significant differences ( > 0.05) regarding protein recovery between both SPE methods. However, only glaucoma-associated marker  showed a significant ( = 0.02) higher abundance in ZIPTIP-purified replicates in comparison to SOLAµ-treated study samples. Nevertheless, this result was not confirmed in the verification experiment using in-gel trypsin digestion of recombinant  ( = 0.24). In conclusion, both SPE-based purification methods worked equally well in terms of analytical performance and reproducibility, whereas the analysis speed and the semi-automation of the SOLAµ spin plates workflow is much more convenient in comparison to the ZIPTIP C18 method.

Article	Year
First insight into the proteome landscape of the porcine short posterior ciliary arteries: Key signalling pathways maintaining physiologic functions. Scientific reports\| PubMed ID: 27922054	2016
Proteomics Unravels the Regulatory Mechanisms in Human Tears Following Acute Renouncement of Contact Lens Use: A Comparison between Hard and Soft Lenses. Scientific reports\| PubMed ID: 30069058	2018
Comparison of Two Solid-Phase Extraction (SPE) Methods for the Identification and Quantification of Porcine Retinal Protein Markers by LC-MS/MS. International journal of molecular sciences\| PubMed ID: 30513899	2018

Natarajan Perumal

Biography

JoVE Journal Publications

Other Publications