A large-scale filter-aided sample preparation method (LFASP) for the analysis of the ubiquitinome

Abstract

1. Introduction Protein ubiquitination regulates key cellular functions, including protein homeostasis and signal transduction. The digestion of ubiquitinated proteins with trypsin yields a glycine-glycine remnant bound to the modified lysine residue (K-¿-GG) that can be recognized by specific antibodies for immunoaffinity purification and subsequent identification of ubiquitination sites by mass spectrometry. Previous ubiquitinome studies based on this strategy have consistently digested milligram amounts of protein as starting material using in-solution digestion protocols prior to K-¿-GG enrichment. Filter-aided sample preparation (FASP) surpasses in-solution protein digestion in cleavage efficiency, but its performance has thus far been shown for digestion of sample amounts on the order of micrograms.

2. Objectives Because cleavage efficiency is pivotal in the generation of the K-¿-GG epitope recognized during immunoaffinity purification, here we aimed to develop a large-scale FASP method (LFASP) for digestion of milligram amounts of protein and evaluate its applicability to the study of the ubiquitinome.

3. Materials & methods Jurkat cells were treated with the proteasome inhibitor Bortezomib. Proteins were digested using LFASP and K-¿-GG peptides were enriched by immunoaffinity purification after peptide fractionation with basic pH reversed-phase liquid chromatography. Peptides were analyzed by LC-MS/MS using an Agilent 1200 HPLC coupled to an LTQ Orbitrap XL mass spectrometer.

4. Results LFASP-based tryptic digestion is efficient (¿90 % of peptides are fully cleaved), robust, reproducible and applicable to the study of the ubiquitinome. We benchmark our results with state-of-the-art ubiquitinome studies and show an ~3-fold reduction in the proportion of miscleaved peptides with the method presented here.

5. Conclusion LFASP enables efficient and reproducible digestion of sample amounts on the order of micrograms and therefore overcomes the general limitation in sample capacity of standard FASP-based protocols. The method is suitable for ubiquitinome analysis and a variety of applications that demand a large(r) amount of starting material.