Large-Scale Quantitative Assessment of Different In-Solution Protein Digestion Protocols Reveals Superior Cleavage Efficiency of Tandem Lys-C/Trypsin Proteolysis over Trypsin Digestion

Abstract

The complete and specific proteolytic cleavage of protein samples into peptides is crucial for the success of every shotgun LC-MS/MS experiment. In particular, popular peptide-based label-free and targeted mass spectrometry approaches rely on efficient generation of fully cleaved peptides to ensure accurate and sensitive protein quantification. In contrast to previous studies, we globally and quantitatively assessed the efficiency of different digestion strategies using a yeast cell lysate, label-free quantification, and statistical analysis. Digestion conditions include double tryptic, surfactant-assisted, and tandem-combinatorial Lys-C/trypsin digestion. In comparison to tryptic digests, Lys-C/trypsin digests were found most efficient to yield fully cleaved peptides while reducing the abundance of miscleaved peptides. Subsequent sequence context analysis revealed improved digestion performances of Lys-C/trypsin for miscleaved sequence stretches flanked by charged basic and particulary acidic residues. Furthermore, targeted MS analysis demonstrated a more comprehensive protein cleavage only after Lys-C/trypsin digestion, resulting in a more accurrate absolute protein quantification and extending the number of peptides suitable for SRM assay development. Therefore, we conclude that a serial Lys-C/trypsin digestion is highly attractive for most applications in quantitative MS-based proteomics building on in-solution digestion schemes.

Quantitative analysis of human centrosome architecture by targeted proteomics and fluorescence imaging

“…The centrosome preparations were analyzed by shotgun proteomics as described previously (Glatter et al , 2012) with minor modifications. The hybrid Orbitrap‐Velos mass spectrometer was interfaced to a nanoelectrospray ion source coupled online to an Easy‐nLC 1000 system (all Thermo Scientific, Waltham, MA, USA).…”

“…The database search results were filtered using the ion score to set the false discovery rate (FDR) to 1% on the peptide and protein level, respectively, based on the number of reverse protein sequence hits in the datasets. After normalizing the quantitative data of the technical replicates using the SafeQuant software tool (Glatter et al , 2012), the summed precursor intensities obtained from Progenesis for each centrosome preparation were aligned with the absolute protein concentrations determined by SRM analysis according to the iBAQ method (Schwanhausser et al , 2011), respectively. The resulting models were applied to estimate absolute protein levels for all proteins quantified in two centrosome preparations, and data were combined by calculating mean protein levels.…”

Analysis of protein–RNA interactions in CRISPR proteins and effector complexes by UV-induced cross-linking and mass spectrometry

“…Some proteomic studies use a first endoproteolytic cleavage step with the enzyme Lys-C, which is still active at higher urea concentrations such as 4 M [39] followed by a second digestion step with trypsin.…”

Exploiting the multiplexing capabilities of tandem mass tags for high-throughput estimation of cellular protein abundances by mass spectrometry

“…Cells were collected by centrifugation at 400g at 4°C, washed twice with 2 ml ice--cold PBS buffer, harvested by centrifugation at 500g and the pellet was snap frozen in liquid nitrogen and stored at --80°C until further processing. For S. pombe, cultures of wild type 972 h+ cells were grown in YE medium [24,30] at 25˚C to concentrations of 3.5x10 6 cells/ml and around 10^8 cells of each sample were collected by centrifugation at 2,000xg, washed twice with PBS buffer and harvested by centrifugation at 2,000xg. Cells were resuspended in 100µl lysis buffer (100 mM ammoniumbicarbonate, 2% sodium deoxycholate, 5mM TCEP), disrupted by strong indirect sonication (100% amplitude, 0.5 cycle, 2 × 10 s) in a Vial Tweeter (Hielscher) and reduced for 15min at 95°C.…”

“…Then, the samples were diluted with 100mM ammoniumbicarbonate buffer to a final sodium deoxycholate concentration of 1% and further digested by incubation with sequencing--grade modified trypsin (1/50, w/w; Promega, Madison, Wisconsin) over night at 37°C. The sequential double Lys--C/trypsin digestion has been recently shown to be more efficient in generating fully cleaved peptides than tryptic digest alone [30] and is therefore recommended for all protein samples analyzed by LC--MS. Sodium deoxycholate is one of a few LC--MS compatible detergents, since it precipitates after acidification [25,33,38] using 2M HCl to a final concentration of 50mM and can then be easily removed by centrifugation at 10,000g for 15min before LC--MS analysis.…”