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 (data-independent 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.

Figure 1.

Outline of the experimental design and the data processing workflow. Organelle separation was performed by density gradient ultracentrifugation. Two biological replicates were acquired. The Golgi apparatus was resolved in the upper part of the gradient, and the corresponding 10 fractions were used for LC-IMS-MS^E data acquisition. The data from all acquisitions were used to reconstruct the protein abundance distribution along the density gradient. The profile data were normalized and used for classification of Golgi proteins versus non-Golgi proteins. The Top-3 quantities from the most enriched fraction for the Golgi apparatus were used to rank order the Golgi-localized proteins.

Figure 2.

Golgi apparatus protein abundance distribution profiles along the density gradients. A, Western-blot analysis of the Golgi marker protein Gtl6-myc. B, Relative protein abundance distribution of Golgi marker proteins along the upper 10 fractions of the density gradient as measured by LC-IMS-MS^E. C, Relative protein abundance distribution of the Golgi-assigned proteins along the upper 10 fractions of the density gradient as measured by LC-IMS-MS^E.

Figure 3.

Rank order distribution of 102 Golgi-localized proteins based on the abundance from Top-3 quantification of the fraction most enriched for the Golgi apparatus. The average value of the two biological replicates is plotted. The ranking of protein abundance forms an S-shaped curve on the logarithmic scale. Protein names referred to in “Discussion” are labeled. The nomenclature of the putative GTs is as in Nikolovski et al. (2012). COBRA is a family of extracellular glycosyl-phosphatidyl inositol-anchored proteins. The data are available in Supplemental Data S5.

Figure 4.

Correlation of Golgi protein abundance (measured by Top-3) between two biological replicates of the fraction most enriched for the Golgi apparatus.