Tandem Digestion Research Articles

Quantitative detection of ricin in beverages using trypsin/Glu-C tandem digestion coupled with ultra-high-pressure liquid chromatography-tandem mass spectrometry.

The toxic protein of ricin has drawn wide attention in recent years as a potential bioterrorism agent due to its high toxicity and wide availability. For the verification of the potential anti-terrorism activities, it is urgent for the quantification of ricin in food-related matrices. Here, a novel strategy of trypsin/Glu-C tandem digestion was introduced for quantitative detection of ricin marker peptides in several beverage matrices using isotope-labeled internal standard (IS)-mass spectrometry. The ricin in beverages was captured and enriched by biotinylated anti-ricin polyclonal antibodies conjugated to streptavidin magnetic beads. The purified ricin was cleaved using the developed trypsin/Glu-C tandem digestion method and then quantitatively detected by ultra-high-pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) with isotope-labeled T7A and TG11B selected as IS.The use of trypsin/Glu-C digestion allows shorter peptides, which are more suitable for MS detection, to be obtained than the use of single trypsin digestion.Under the optimized tandem digestion condition, except for T7A in the A-chain, two resulting specific peptides of TG13A, TG28A from the A-chain and two of TG11B, TG33B from the B-chain were chosen as novel marker peptides with high MS response. The uniqueness of the selected marker peptides allows for unambiguous identification of ricin among its homologous proteins in a single run. The MS response of the four novel marker peptides is increased by more than 10 times compared with that of individual corresponding tryptic peptides. Both the marker peptides of A-chain T7A and B-chain TG11B were selected as quantitative peptides based on the highest MS response among the marker peptides from their individual chains. The limit of detection (LOD) of ricin is 0.1ng/mL in PBS and 0.5ng/mL in either milk or orange juice. The linear range of calibration curves for ricin were 0.5-300ng/mL in PBS, 1.0-400ng/mL in milk, and 1.0-250ng/mL in orange juice. The method accuracy ranged between 82.6 and 101.8% for PBS, 88.9-105.2% for milk, and 95.3-118.7% for orange juice. The intra-day and inter-day precision had relative standard deviations (%RSD) of 0.3-9.4%, 0.7-8.9%, and 0.2-6.9% in the three matrices respectively. Furthermore, whether T7A or TG11B is used as a quantitative peptide, the quantitative results of ricin are consistent. This study provides not only a practical method for the absolute quantification of ricin in beverage matrices but also a new strategy for the investigation of illegal use of ricin in chemical weapon verification tasks such as OPCW biotoxin sample analysis exercises.

Ac-LysargiNase Complements Trypsin for the Identification of Ubiquitinated Sites.

Mass spectrometry (MS)-based identification of ubiquitinated sites requires trypsin digestion prior to MS analysis, and a signature peptide was produced with a diglycine residue attached to the ubiquitinated lysine (K-ε-GG peptide). However, the missed cleavage of modified lysines by trypsin results in modified peptides with increased length and charge, whose detection by MS analysis is suppressed by the vast majority of internally unmodified peptides. LysargiNase, the mirrored trypsin, is reported to cleave before lysine and arginine residues and to be favorable for the identification of methylation and phosphorylation, but its digestive characteristics related to ubiquitination are unclear. Herein, we tested the capacity of the in-house developed acetylated LysargiNase (Ac-LysargiNase) with high activity and stability, for cleaving ubiquitinated sites in both the seven types of ubiquitin chains and their corresponding K-ε-GG peptides. Interestingly, Ac-LysargiNase could efficiently cleave the K63-linked chain but had little effect on the other types of chains. Additionally, Ac-LysargiNase had higher exopeptidase activity than trypsin. Utilizing these features of the paired mirror proteases, a workflow of trypsin and Ac-LysargiNase tandem digestion was developed for the identification of ubiquitinated proteins. Through this method, the charge states and ionization capacity of the unmodified peptides were efficiently reduced, and the identification of modified sites was consequently increased by 30% to 50%. Strikingly, approximately 15% of the modified sites were cleaved by Ac-LysargiNase, resulting in shorter K-ε-GG peptides for better identification. The enzyme Ac-LysargiNase is expected to serve as an option for increasing the efficiency of modified site identification in ubiquitome research.

Efficient Tandem LysC/Trypsin Digestion in Detergent Conditions.

All shotgun proteomics experiments rely on efficient proteolysis steps for sensitive peptide/protein identification and quantification. Previous reports suggest that the sequential tandem LysC/trypsin digest yields higher recovery of fully tryptic peptides than single-tryptic proteolysis. Based on the previous studies, it is assumed that the advantageous effect of tandem proteolysis requires a high sample denaturation state for the initial LysC digest. Therefore, to date, all systematic assessments of LysC/trypsin proteolysis are done in chaotropic environments such as urea. Here, sole trypsin is compared with LysC/trypsin and it is shown that tandem digestion can be carried with high efficiency in Mass Spectrometry-compatible detergents, thereby resulting in higher quantitative yields of fully cleaved peptides. It is further demonstrated that higher cleavage efficiency of tandem digests has a positive impact on absolute protein quantification using intensity-based absolute quantification (iBAQ) values. The results of the examination of divergent urea tandem conditions imply that beneficial effects of the initial LysC digest do not depend on the sample denaturation state, but, are mainly caused by different target specificities of LysC and trypsin. The observed detergent compatibility enables tandem digestion schemes to be implemented in efficient cellular solubilization proteomics procedures without the need for buffer exchange to chaotropic environments.

LC-HRMS Screening and Identification of Novel Peptide Markers of Ricin Based on Multiple Protease Digestion Strategies.

Both ricin and R. communis agglutinin (RCA120), belonging to the type II ribosome-inactivating proteins (RIPs-Ⅱ), are derived from the seeds of the castor bean plant. They share very similar amino acid sequences, but ricin is much more toxic than RCA120. It is urgently necessary to distinguish ricin and RCA120 in response to public safety. Currently, mass spectrometric assays are well established for unambiguous identification of ricin by accurate analysis of differentiated amino acid residues after trypsin digestion. However, diagnostic peptides are relatively limited for unambiguous identification of trace ricin, especially in complex matrices. Here, we demonstrate a digestion strategy of multiple proteinases to produce novel peptide markers for unambiguous identification of ricin. Liquid chromatography-high resolution MS (LC-HRMS) was used to verify the resulting peptides, among which only the peptides with uniqueness and good MS response were selected as peptide markers. Seven novel peptide markers were obtained from tandem digestion of trypsin and endoproteinase Glu-C in PBS buffer. From the chymotrypsin digestion under reduction and non-reduction conditions, eight and seven novel peptides were selected respectively. Using pepsin under pH 1~2 and proteinase K digestion, six and five peptides were selected as novel peptide markers. In conclusion, the obtained novel peptides from the established digestion methods can be recommended for the unambiguous identification of ricin during the investigation of illegal use of the toxin.

Lys-C/Trypsin Tandem-Digestion Protocol for Gel-Free Proteomic Analysis of Colon Biopsies.

The protocol presented was specifically optimized for in-depth analysis of the human colon mucosa proteome. After cell lysis in a sodium deoxycholate/urea buffer, a tandem digestion with Lys-C and trypsin was performed. Prior to LC-MS/MS analysis, peptides were TMT-labeled and fractionated by high pH reversed-phase spin columns. This protocol is a powerful, reproducible, sample-saving, and cost-effective option when an in-depth quantitative proteome analysis is desired.

Lys-C/Arg-C, a More Specific and Efficient Digestion Approach for Proteomics Studies.

Nowadays, bottom-up approaches are predominantly adopted in proteomics studies, which necessitate a proteolysis step prior to MS analysis. Trypsin is often the best protease in choice due to its high specificity and MS-favored proteolytic products. A lot of efforts have been made to develop a superior digestion approach but hardly succeed, especially in large-scale proteomics studies. Herein, we report a new tandem digestion using Lys-C and Arg-C, termed Lys-C/Arg-C, which has been proven to be more specific and efficient than trypsin digestion. Reanalysis of our previous data ( Anal. Chem. 2018, 90 (3), 1554-1559) revealed that both Lys-C and Arg-C are trypsin-like proteases and perform better when considered as trypsin. In particular, for Arg-C, the identification capacity is increased to 2.6 times and even comparable with trypsin. The good complementarity, high digestion efficiency, and high specificity of Lys-C and Arg-C prompt the Lys-C/Arg-C digestion. We systematically evaluated Lys-C/Arg-C digestion using qualitative and quantitative proteomics approaches and confirmed its superior performance in digestion specificity, efficiency, and identification capacity to the currently widely used trypsin and Lys-C/trypsin digestions. As a result, we concluded that the Lys-C/Arg-C digestion approach would be the choice of next-generation digestion approach in both qualitative and quantitative proteomics studies. Data are available via ProteomeXchange with identifier PXD009797.

The Proteome of Ulcerative Colitis in Colon Biopsies from Adults - Optimized Sample Preparation and Comparison with Healthy Controls.

The purpose of the study was to optimize the sample preparation and to further use an improved sample preparation to identify proteome differences between inflamed ulcerative colitis tissue from untreated adults and healthy controls. To optimize the sample preparation, we studied the effect of adding different detergents to a urea containing lysis buffer for a Lys-C/trypsin tandem digestion. With the optimized method, we prepared clinical samples from six ulcerative colitis patients and six healthy controls and analysed them by LC-MS/MS. We examined the acquired data to identify differences between the states. We improved the protein extraction and protein identification number by utilizing a urea and sodium deoxycholate containing buffer. Comparing ulcerative colitis and healthy tissue, we found 168 of 2366 identified proteins differently abundant. Inflammatory proteins are higher abundant in ulcerative colitis, proteins related to anion-transport and mucus production are lower abundant. A high proportion of S100 proteins is differently abundant, notably with both up-regulated and down-regulated proteins. The optimized sample preparation method will improve future proteomic studies on colon mucosa. The observed protein abundance changes and their enrichment in various groups improve our understanding of ulcerative colitis on protein level.

Investigation of bi-enzymatic reactor based on hybrid monolith with nanoparticles embedded and its proteolytic characteristics

The bottom-up strategy of proteomic profiling study based on mass spectrometer (MS) has drawn high attention. However, conventional solution-based digestion could not satisfy the demands of highly efficient and complete high throughput proteolysis of complex samples. We proposed a novel bi-enzymatic reactor by immobilizing two different enzymes (trypsin/chymotrypsin) onto a mixed support of hybrid organic–inorganic monolith with SBA-15 nanoparticles embedded. Typsin and chymotrypsin were crossly immobilized onto the mixed support by covalent bonding onto the monolith with glutaraldehyde as bridge reagent and chelation via copper ion onto the nanoparticles, respectively. Compared with single enzymatic reactors, the bi-enzymatic reactor improved the overall functional analysis of membrane proteins of rat liver by doubling the number of identified peptides (from 1184/1010 with trypsin/chymotrypsin enzymatic reactors to 2891 with bi-enzymatic reactor), which led to more proteins identified with deep coverage (from 452/336 to 620); the efficiency of the bi-enzymatic reactor is also better than that of solution-based tandem digestion, greatly shorting the digestion time from 24h to 50s. Moreover, more transmembrane proteins were identified by bi-enzymatic reactor (106) compared with solution-based tandem digestion (95) with the same two enzymes and enzymatic reactors with single enzyme immobilized (75 with trypsin and 66 with chymotrypsin). The proteolytic characteristics of the bi-enzymatic reactors were evaluated by applying them to digestion of rat liver proteins. The reactors showed good digestion capability for proteins with different hydrophobicity and molecular weight.

An ultra-fast and highly efficient multiple proteases digestion strategy using graphene-oxide-based immobilized protease reagents

Highly efficient and rapid proteolytic digestion of proteins into peptides is a crucial step in shotgun-based proteome-analysis strategy. Tandem digestion by two or more proteases is demonstrated to be helpful for increasing digestion efficiency and decreasing missed cleavages, which results in more peptides that are compatible with mass-spectrometry analysis. Compared to conventional solution digestion, immobilized protease digestion has the obvious advantages of short digestion time, no self-proteolysis, and reusability. We proposed a multiple-immobilized proteases-digestion strategy that combines the advantages of the two digestion strategies mentioned above. Graphene-oxide (GO)-based immobilized trypsin and endoproteinase Glu-C were prepared by covalently attaching them onto the GO surface. The prepared GO-trypsin and GO-Glu-C were successfully applied in standard protein digestion and multiple immobilized proteases digestion of total proteins of Thermoanaerobacter tengcongensis. Compared to 12-hour solution digestion using trypsin or Glu-C, 14% and 7% improvement were obtained, respectively, in the sequence coverage of BSA by one-minute digestion using GO-trypsin and GO-Glu-C. Multiple immobilized-proteases digestion of the total proteins of Thermoanaerobacter tengcongensis showed 24.3% and 48.7% enhancement in the numbers of identified proteins than was obtained using GO-trypsin or GO-Glu-C alone. The ultra-fast and highly efficient digestion can be contributed to the high loading capacity of protease on GO, which leads to fewer missed cleavages and more complete digestion. As a result, improved protein identification and sequence coverage can be expected.

Improve the Coverage for the Analysis of Phosphoproteome of HeLa Cells by a Tandem Digestion Approach

Complete coverage of all phosphorylation sites in a proteome is the ultimate goal for large-scale phosphoproteome analysis. However, only making use of one protease trypsin for protein digestion cannot cover all phosphorylation sites, because not all tryptic phosphopeptides are detectable in MS. To further increase the phosphoproteomics coverage of HeLa cells, we proposed a tandem digestion approach by using two different proteases. By combining the data set of the first Glu-C digestion and the second trypsin digestion, the tandem digestion approach resulted in the identification of 8062 unique phosphopeptides and 8507 phosphorylation sites in HeLa cells. The conventional trypsin digestion approach resulted in the identification of 3891 unique phosphopeptides and 4647 phosphorylation sites. It was found that the phosphorylation sites identified from the above two approaches were highly complementary. By combining above two data sets, in total we identified 10899 unique phosphopeptides and 11262 phosphorylation sites, corresponding to 3437 unique phosphoproteins with FDR < 1% at peptide level. We also compared the kinase motifs extracted from trypsin, Glu-C, or a second trypsin digestion data sets. It was observed that basophilic motifs were more frequently found in the trypsin and the second trypsin digestion data sets, and the acidic motifs were more frequently found in the Glu-C digestion data set. These results demonstrated that our tandem digestion approach is a good complement to the conventional trypsin digestion approach for improving the phosphoproteomics analysis coverage of HeLa cells.

An improved method for genome wide DNA methylation profiling correlated to transcription and genomic instability in two breast cancer cell lines

BackgroundDNA methylation is a widely studied epigenetic mechanism known to correlate with gene repression and genomic stability. Development of sensitive methods for global detection of DNA methylation events is of particular importance.ResultsWe here describe a technique, called modified methylation-specific digital karyotyping (MMSDK) based on methylation-specific digital karyotyping (MSDK) with a novel sequencing approach. Briefly, after a tandem digestion of genomic DNA with a methylation-sensitive mapping enzyme and a fragmenting enzyme, short sequence tags are obtained. These tags are amplified, followed by direct, massively parallel sequencing (Solexa 1G Genome Analyzer). This method allows high-throughput and low-cost genome-wide DNA methylation mapping. We applied this method to investigate global DNA methylation profiles for widely used breast cancer cell lines, MCF-7 and MDA-MB-231, which are representatives for luminal-like and mesenchymal-like cancer types, respectively. By comparison, a highly similar overall DNA methylation pattern was revealed for the two cell lines. However a cohort of individual genomic loci with significantly different DNA methylation status between two cell lines was identified. Furthermore, we revealed a genome-wide significant correlation between gene expression and the methylation status of gene promoters with CpG islands (CGIs) in the two cancer cell lines, and a correlation of gene expression and the methylation status of promoters without CGIs in MCF-7 cells.ConclusionThe MMSDK method will be a valuable tool to increase the current knowledge of genome wide DNA methylation profiles.