Absolute Quantification of Proteins in the Eye of Drosophila melanogaster

Abstract

Absolute (molar) quantification of proteins determines their molar ratios in complexes, networks, and metabolic pathways. MS Western workflow is employed to determine molar abundances of proteins potentially critical for morphogenesis and phototransduction (PT) in eyes of Drosophila melanogaster using a single chimeric 264 kDa protein standard that covers, in total, 197 peptides from 43 proteins. The majority of proteins are independently quantified with two to four proteotypic peptides with the coefficient of variation of less than 15%, better than 1000-fold dynamic range and sub-femtomole sensitivity. Here, the molar abundance of proteins of the PT machinery and of the rhabdomere, the photosensitive organelle, is determined in eyes of wild-type flies as well as in crumbs (crb) mutant eyes, which exhibit perturbed rhabdomere morphogenesis.

Hydroxylated sphingolipid biosynthesis regulates photoreceptor apical domain morphogenesis

“…In the distal regions, the rhabdomeres appear wider in crb 8F105 (L), and Increased fa2h levels limit Rh1 delivery to rhabdomeres Besides improperly expanded and bulky rhabdomeres, punctate cytoplasmic accumulation of Rh1 is another feature of the crb mutant phenotype (Pocha et al, 2011), pointing to impaired Rh1 trafficking. As recently shown, absolute levels of opsin do not change in crb mutants with bulky rhabdomeres (Raghuraman et al, 2020). To further understand whether up-regulated fa2h impacts Rh1 distribution as well, we analyzed the localization of Rh1 by immunostaining.…”

Median based absolute quantification of proteins using Fully Unlabelled Generic Internal Standard (FUGIS)

“…In order to assess whether using BestN peptides delivered better accuracy, we looked into the quantification of one of the four proteins (glyceraldyhyde-3 phosphate dehydrogenase (G3P Human P04406)) (Figure 5A) (Supplementary information, MBAQ vs MSW vs Hi3). We estimated the concordance of the molar amount independently calculated from multiple peptides by the coefficient of variation [9] . The coefficient of variation (%CV) of the molar amounts calculated using BestN peptides was 7 %, which was much lesser than that of the Hi-3 quantification (18 %) (Figure 5B) (Figure S3) (Supplementary information, MBAQ vs MSW vs Hi3).…”

“…The chromatographic alignment and feature detection were carried using Progenesis LC-MS v.4.1 (Nonlinear Dynamics, UK). The absolute quantification was performed by calculating the abundances for the labelled and the unlabelled peptide using an in-house software as previously published [9] . The statistical analysis were carried out in OriginLab (2017) (OriginLab Corp., Northampton, Massachusetts, USA).…”

“…The peptide matching and chromatographic peak alignment from the raw files was carried out as described in (Supplementary information, Database search and data processing). The quantification was performed using the software developed in-house 9 .…”

“…In contrast, absolute quantification determines the exact molar quantity of each protein in each condition. In this way, it is possible to relate the molar abundances of different proteins, estimate their expression level or determine their stoichiometry within a variety of molecular constellations from stable complexes to organelles or metabolic pathways and interaction networks [1][2][3][4][5][6][7][8][9][10] . Absolute quantification holds an important promise to deliver reference values of individual proteins in liquid and solid biopsies, which is a pre-requisite for robust molecular diagnostics.…”