Label-Free Protein Quantification for Plant Golgi Protein Localization and Abundance

Abstract

The proteomic composition of the Arabidopsis (Arabidopsis thaliana) Golgi apparatus is currently reasonably well documented; however, little is known about the relative abundances between different proteins within this compartment. Accurate quantitative information of Golgi resident proteins is of great importance: it facilitates a better understanding of the biochemical processes that take place within this organelle, especially those of different polysaccharide synthesis pathways. Golgi resident proteins are challenging to quantify because the abundance of this organelle is relatively low within the cell. In this study, an organelle fractionation approach targeting the Golgi apparatus was combined with a label-free quantitative mass spectrometry (dataindependent acquisition method using ion mobility separation known as LC-IMS-MS E [or HDMS E ]) to simultaneously localize proteins to the Golgi apparatus and assess their relative quantity. In total, 102 Golgi-localized proteins were quantified. These data show that organelle fractionation in conjunction with label-free quantitative mass spectrometry is a powerful and relatively simple tool to access protein organelle localization and their relative abundances. The findings presented open a unique view on the organization of the plant Golgi apparatus, leading toward unique hypotheses centered on the biochemical processes of this organelle.

MUCILAGE-RELATED10 Produces Galactoglucomannan That Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage

“…11, G-I). These results are consistent with MUCI10/GTL6 serving as a Golgi marker in multiple proteomic studies (Dunkley et al, 2004(Dunkley et al, , 2006Nikolovski et al, 2012Nikolovski et al, , 2014.…”

“…11; Nikolovski et al, 2014;Yu et al, 2014) and are likely the key enzymes required for GGM synthesis in the Golgi apparatus. Although we did not detect in vitro galactosyltransferase activity for MUCI10 recombinant proteins purified from E. coli or expressed in N. benthamiana microsomes, only a few plant glycosyltransferases have been biochemically characterized through direct assays.…”

“…MUCI10/GT7 (At2g22900) and its paralog GT6 (At4g37690) do not function as XXTs (Vuttipongchaikij et al, 2012) and are more closely related to the TfGMGT enzyme (Faik et al, 2002;Keegstra and Cavalier, 2010). MUCI10, also called GALACTOSYLTRANSFERASE-LIKE6 (GTL6), served as a Golgi marker in multiple proteomic studies of Arabidopsis callus cultures (Dunkley et al, 2004(Dunkley et al, , 2006Nikolovski et al, 2012Nikolovski et al, , 2014. Nevertheless, the role of TfGMGT orthologs in Arabidopsis remained unknown.…”

Multidimensional proteomics for cell biology

“…An advantage of DIA strategies is the ability to systematically analyse the peptide population in an extract, which facilitates a more consistent quantitative analysis across large numbers of samples. DIA has recently been used for example, to characterize changes in the macrophage proteome in response to HIV-1 infection 27 , the cyclin-dependent kinase 4 interactom e 28 and the plant Golgi apparatus 29 .…”

Comparative “Golgi” Proteome Study of Lolium multiflorum and Populus trichocarpa

“…There has been some progress towards mapping the GA proteome in the model plant system Arabidopsis [6,7,8,9,10]. However, it has been estimated that out of the 2239 ± 465 predicted GA proteins only 20% have been experimentally identified thus far by combining all published Arabidopsis GA proteomes [2].…”