Understanding the Role of Proteolytic Digestion on Discovery and Targeted Proteomic Measurements Using Liquid Chromatography Tandem Mass Spectrometry and Design of Experiments

Abstract

Workflows in bottom-up proteomics have traditionally implemented the use of proteolysis during sample preparation; enzymatic digestion is most commonly performed using trypsin. This results in the hydrolysis of peptide bonds forming tryptic peptides, which can then be subjected to LC-MS/MS analysis. While the structure, specificity, and kinetics of trypsin are well characterized, a lack of consensus and understanding has remained regarding fundamental parameters critical to obtaining optimal data from a proteomics experiment. These include the type of trypsin used, pH during digestion, incubation temperature as well as enzyme-to-substrate ratio. Through the use of design of experiments (DOE), we optimized these parameters, resulting in deeper proteome coverage and a greater dynamic range of measurement. The knowledge gained from optimization of a discovery-based proteomics experiment was applied to targeted LC-MS/MS experiments using protein cleavage-isotope dilution mass spectrometry for absolute quantification. We demonstrated the importance of these digest parameters with respect to our limit of detection as well as our ability to acquire more accurate quantitative measurements. Additionally, we were able to quantitatively account for peptide decay observed in previous studies, caused by nonspecific activity of trypsin. The tryptic digest optimization described here has eliminated this previously observed peptide decay as well as provided a greater understanding and standardization for a common but critical sample treatment used across the field of proteomics.

Optimizing Mass Spectrometry Analyses: A Tailored Review on the Utility of Design of Experiments

“…Optimization of digestions has been performed previously to maximize protein coverage [9], but protein complexes pose unique challenges and potentially called for establishment of new parameters. As the factors affecting tryptic enzymatic activity had been established (pH, time, temperature), this offered an opportunity to perform a focused response surface study to determine optimal settings.…”

Establishing ion ratio thresholds based on absolute peak area for absolute protein quantification using protein cleavage isotope dilution mass spectrometry

“…Increasing sample complexity and scale has put more demand on instruments and investigators to provide high-throughput quantification. This demand has required optimization of targeted LC-MS/MS methods in order to allow data to be collected and analyzed rapidly and reliably 6,7 . An obstacle that has become a major concern in targeted proteomics is the confidence in the specificity by which a peptide or other biological molecule is identified as well as the accuracy and precision of the absolute value obtained for a given experiment.…”

Multiplexed assay for protein quantitation in the invertebrate Gammarus fossarum by liquid chromatography coupled to tandem mass spectrometry

“…Proteins can differ greatly in their susceptibility to proteolysis, and this requires a digestion protocol that is optimal for all proteins for accurate measurement. The optimal digestion protocol has been shown to be analytedependent and may be unattainable for all proteins if a single digestion protocol is used [23]. To determine the optimal digestion conditions, we first evaluated the total protein concentration used for tryptic digestion and adjusted the trypsin-tosubstrate ratio.…”

“…The modulation of the two other proteins is in accordance with what is known about their potential functional role in crustaceans. PPO is the precursor form of the enzyme phenoloxidase (PO), which participates in the molting process of crustaceans [23,24]. A study in the crustacean decapod Macrobrachium rosenbergii [25] has shown that the expression of the PPO gene reaches a peak during the post-molt stages, and is significantly decreased during the inter-and pre-molt stages.…”