Large-Scale Filter-Aided Sample Preparation Method for the Analysis of the Ubiquitinome

Abstract

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-ε-GG) that can be recognized by specific antibodies for immunoaffinity purification (IAP) 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-ε-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. Because cleavage efficiency is pivotal in the generation of the K-ε-GG epitope recognized during IAP, here we developed a large-scale FASP method (LFASP) for digestion of milligram amounts of protein and evaluated its applicability to the study of the ubiquitinome. Our results demonstrate that LFASP-based tryptic digestion is efficient, robust, reproducible, and applicable to the study of the ubiquitinome. We benchmark our results with state-of-the-art ubiquitinome studies and show a ∼3-fold reduction in the proportion of miscleaved peptides with the method presented here. Beyond ubiquitinome analysis, LFASP overcomes the general limitation in sample capacity of standard FASP-based protocols and can therefore be used for a variety of applications that demand a large(r) amount of starting material.

A Comprehensive Tyrosine Phosphoproteomic Analysis Reveals Novel Components of the Platelet CLEC-2 Signaling Cascade

“…After that, 800 µg of protein per sample were digested with trypsin following a large-scale filter-aided sample preparation method on 10 kDa filters (Amicon Ultra-15, Millipore). 16 Tryptic digests were dried and resuspended in 700 µL of IAP buffer (50 mM MOPS pH 7.2; 10 mM disodium phosphate; 50 mM sodium chloride).…”

Discovery of large molecules as new biomarkers in wastewater using environmental proteomics and suitable polymer probes

“…Separation was carried out at 400 nL/min using a 60-min linear gradient from 0 to 35 % solvent B Journal Pre-proof J o u r n a l P r e -p r o o f 6 (Solvent A: H2O, 0.1 % (v/v) formic acid; solvent B: ACN, 0.1 % (v/v) formic acid). The LTQ Orbitrap XL was operated in data-dependent mode (Casanovas et al, 2017).…”

Evaluation of FASP, SP3, and iST Protocols for Proteomic Sample Preparation in the Low Microgram Range

“…The proteolytically generated peptides are small enough to pass the filter and can be collected by centrifugation. Over the past years, FASP has seen widespread utility for sample processing and has been adapted for many different applications including the analysis of different cell and tissue types, − formalin-fixed paraffin-embedded tissues, , the large-scale characterization of the ubiqitinome, mapping of the N -glycoproteome, as well as the brain phosphoproteome . Typically, after FASP digestion, about 50% of starting material can be recovered, which has no adverse effects on proteome coverage when higher amounts of sample are processed. ,, However, incomplete peptide recovery after FASP digestion can be a problem when processing only minute amounts of material.…”