Trypsin Digestion Of Proteins Research Articles

Optimization of the linear quantification range of an online trypsin digestion coupled liquid chromatography–tandem mass spectrometry (LC–MS/MS) platform

ABSTRACTTandem mass spectrometry (MS/MS)-based proteomic workflows with a bottom-up approach require enzymatic digestion of proteins to peptide analytes, usually by trypsin. Online coupling of trypsin digestion of proteins, using an immobilized enzyme reactor (IMER), with liquid chromatography (LC) and MS/MS is becoming a frequently used approach. However, finding IMER digestion conditions that allow quantitative analysis of multiple proteins with wide range of endogenous concentration requires optimization of multiple interactive parameters: digestion buffer flow rate, injection volume, sample dilution, and surfactant type/concentration. In this report, we present a design of experiment approach for the optimization of an integrated IMER-LC–MS/MS platform. With bovine serum albumin as a model protein, the digestion efficacy and digestion rate were monitored based on LC–MS/MS peak area count versus protein concentration regression. The optimal parameters were determined through multivariate surface response modeling and consideration of diffusion controlled immobilized enzyme kinetics. The results may provide guidance to other users for the development of quantitative IMER-LC–MS/MS methods for other proteins.

Abstract 3871: Simultaneous absolute quantitation of ATP-binding cassette transporters in canine tissues via targeted proteomics

Abstract The dog serves as a useful model of spontaneously occurring cancer by virtue of similar biology, therapies and mechansims of chemotherapeutic resistance. The ABC transporters are a superfamily of membrane bound proteins that are widely distributed across mammalian tissues and constitute a substantial component of variability in anticancer drug pharmakokinetics and tumor cell resistance. Thus a quantitative atlas of ATP-binding cassette (ABC) transporters across canine tissues is crucial for improving our ability to extrapolate data from dog to human. We have employed a quantitative proteomic approach based on multiple reaction monitoring in tandem mass spectrometry that relies on detection of novel peptide sequences following trypsin digestion of biological samples and separation of peptides utilizing high-pressure liquid chromatography. The 14 proteins evaluated include P-gp, Bcrp, Bsep, Mrp1-6, Mrp7, Mrp9, and the products of canine Abcg5 and Abcg8. Peptide sequences were identified via an in-silico trypsin digestion of proteins and signature peptides were generated for identification and quantitation using stable isotope-labeled signature peptides as an internal reference. Signature peptides were chosen based on lack of posttranslational modification sites and known polymorphisms. Isolated brain microvessels, liver, kidney, duodenum, jejunum, ileum and lung were collected at necropsy from ten normal dogs and membrane fractions of each tissue were prepared. Additional samples were collected for isolation of mRNA. Membrane protein fractions were digested with trypsin and total protein concentrations determined by BCA assay. Calibration curves for signature peptides were prepared in a bovine serum albumin digestate and ranged from 0.25 fmol to 200 fmol on column. Quantitation of peptides in tissue samples was performed via linear regression analysis of calibration curves with extrapolation using at least two transitions for each peptide. Results were obtained as femtomole of peptide per microgram of total protein in the sample. Differential protein levels across tissues were compared to quantitative real time RT-PCR results for the corresponding genes and differences between tissues with each method were concordant. Our results provide the first quantitative atlas of ABC transporters across normal dog tissues and provide a framework for improved understanding of pharmacokinetic and pharmacodynamic variability among dogs receiving anticancer therapies. In addition, this panel will allow evaluation of the potential role that each of these transporters plays in resistance to chemotherapy in vitro and in vivo in dogs, a relevant preclinical model of naturally occurring cancers. Citation Format: Luke A. Wittenburg, Holly Conger, Dominique Ramirez, Daniel Gustafson. Simultaneous absolute quantitation of ATP-binding cassette transporters in canine tissues via targeted proteomics. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3871.

Measurement of glycated albumin in serum and plasma by LC-MS/MS

Background Diagnosis of diabetes and monitoring of long-term blood sugar are preferably done by measurement of glycated hemoglobin (HbA1c). Diabetic patients with end stage renal disease (ESRD) may have short-lived red blood cells due to hemodialysis (HD), and thus higher turnover of hemoglobin. The level of glycated hemoglobin (HbA1c) may be lower than expected for these patients, even at increased blood glucose, possibly making glycated albumin (GA) measurement a better alternative. Methods The percentage of GA was measured by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Fast and efficient trypsin digestion of proteins in diluted serum or plasma resulted in a high number of proteotypic peptides from albumin, including KQTALVELVK which was detected both glycated and non-glycated by multiple reaction monitoring (MRM). The percentage of GA was estimated by neat peak area response of glycated peptide divided by the sum of glycated and non-glycated peptide. Results Acceptable method reproducibility (6% CV), repeatability (2–6% CV), limit of quantification (0.75% GA), linearity (R2 = 0.999) and recovery (79 ± 9%) was achieved without using calibration or isotope-labeled internal standard. GA was strongly correlated with HbA1c (r = 0.84) for patients without ESRD. The average ratio of GA/HbA1c was significantly higher (p = 0.0021) for ESRD patients (1.84 ± 0.38, n = 62) compared to other patients (1.67 ± 0.28, n = 225). Conclusion GA measurement by detecting glycation in KQTALVELVK with LC-MS/MS seems to be a useful supplement to HbA1c for detecting increased blood glucose in diabetic patients with ESRD.

Rapid and Accurate Identification of Animal Species in Natural Leather Goods by Liquid Chromatography/Mass Spectrometry.

The demand for leather goods has grown globally in recent years. Industry revenue is forecast to reach $91.2 billion by 2018. There is an ongoing labelling problem in the leather items market, in that it is currently impossible to identify the species that a given piece of leather is derived from. To address this issue, we developed a rapid and simple method for the specific identification of leather derived from cattle, horses, pigs, sheep, goats, and deer by analysing peptides produced by the trypsin-digestion of proteins contained in leather goods using liquid chromatography/mass spectrometry. We determined species-specific amino acid sequences by liquid chromatography/tandem mass spectrometry analysis using the Mascot software program and demonstrated that collagen α-1(I), collagen α-2(I), and collagen α-1(III) from the dermal layer of the skin are particularly useful in species identification.

Large-scale identification of ubiquitination sites by mass spectrometry

Ubiquitination is essential for the regulation of cellular protein homeostasis. It also has a central role in numerous signaling events. Recent advances in the production and availability of antibodies that recognize the Lys-ɛ-Gly-Gly (K-ɛ-GG) remnant produced by trypsin digestion of proteins having ubiquitinated lysine side chains have markedly improved the ability to enrich and detect endogenous ubiquitination sites by mass spectrometry (MS). The following protocol describes the steps required to complete a large-scale ubiquitin experiment for the detection of tens of thousands of distinct ubiquitination sites from cell lines or tissue samples. Specifically, we present detailed, step-by-step instructions for sample preparation, off-line fractionation by reversed-phase chromatography at pH 10, immobilization of an antibody specific to K-ɛ-GG to beads by chemical cross-linking, enrichment of ubiquitinated peptides using these antibodies and proteomic analysis of enriched samples by LC-tandem MS (MS/MS). Relative quantification can be achieved by performing stable isotope labeling by amino acids in cell culture (SILAC) labeling of cells. After cell or tissue samples have been prepared for lysis, the described protocol can be completed in ∼5 d.

Perfluorooctanoic Acid for Shotgun Proteomics

Here, we describe the novel use of a volatile surfactant, perfluorooctanoic acid (PFOA), for shotgun proteomics. PFOA was found to solubilize membrane proteins as effectively as sodium dodecyl sulfate (SDS). PFOA concentrations up to 0.5% (w/v) did not significantly inhibit trypsin activity. The unique features of PFOA allowed us to develop a single-tube shotgun proteomics method that used all volatile chemicals that could easily be removed by evaporation prior to mass spectrometry analysis. The experimental procedures involved: 1) extraction of proteins in 2% PFOA; 2) reduction of cystine residues with triethyl phosphine and their S-alkylation with iodoethanol; 3) trypsin digestion of proteins in 0.5% PFOA; 4) removal of PFOA by evaporation; and 5) LC-MS/MS analysis of the resulting peptides. The general applicability of the method was demonstrated with the membrane preparation of photoreceptor outer segments. We identified 75 proteins from 1 µg of the tryptic peptides in a single, 1-hour, LC-MS/MS run. About 67% of the proteins identified were classified as membrane proteins. We also demonstrate that a proteolytic 18O labeling procedure can be incorporated after the PFOA removal step for quantitative proteomic experiments. The present method does not require sample clean-up devices such as solid-phase extractions and membrane filters, so no proteins/peptides are lost in any experimental steps. Thus, this single-tube shotgun proteomics method overcomes the major drawbacks of surfactant use in proteomic experiments.

Application of a temperature-controllable microreactor to simple and rapid protein identification using MALDI-TOF MS

We have carried out a simultaneous thermal denaturation and trypsin digestion of proteins using a temperature-controllable microreactor. This is a simple and rapid sample preparation technique for use before matrix-assisted laser desorption ionization time-of-flight mass spectrometry. In contrast to a conventional sample preparation method, which involves several chemical treatments, our sample preparation was performed using only trypsin digestion with the thermal denaturation of the target protein. Optimization of the reactor operational parameters for trypsin digestion using a temperature-controllable microreactor was carried out. The entire trypsin digestion procedure took about 11 min, and consisted of 1 min for the thermal denaturation of the sample protein (3 microl, 0.2 microM) at 85 degrees C, and 10 min for digestion of the protein at 37 degrees C. The resulting sequence coverage ranged from 24% to 57%, which was sufficient for practical protein identification.

Characterization of Enterobacteria Using MALDI-TOF Mass Spectrometry

A method is proposed for the rapid classification of Gram-negative Enterobacteria using on-slide solubilization and trypsin digestion of proteins, followed by MALDI-TOF MS analysis. Peptides were identified from tryptic digests using microsequencing by tandem mass spectrometry and database searches. Proteins from the outer membrane family (OMP) were consistently identified in the Enterobacteria Escherichia coli, Enterobacter cloacae, Erwinia herbicola, and Salmonella typhimurium. Database searches indicate that these OMP peptides observed are unique to the Enterobacteria order.

Multistep microreactions with proteins using electrocapture technology.

A method to perform multistep reactions by means of electroimmobilization of a target molecule in a microflow stream is presented. A target protein is captured by the opposing effects between the hydrodynamic and electric forces, after which another medium is injected into the system. The second medium carries enzymes or other reagents, which are brought into contact with the target protein and react. The immobilization is reversed by disconnecting the electric field, upon which products are collected at the outlet of the device for analysis. On-line reduction, alkylation, and trypsin digestion of proteins is demonstrated and was monitored by MALDI mass spectrometry.

Trypsin digestion of proteins on intact immobilized pH gradient strips for surface matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis.

Isolelectric focusing (IEF) of proteins on immobilized pH gradient (IPG) strips is an integral part of two-dimensional (2D) electrophoresis-based proteomics. Proteins can be effectively analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) on the intact strip itself, leading to the creation of a virtual 2D map giving pI and MW information, bypassing the second dimension SDS-PAGE. Further, trypsin digestion of proteins on the strip can significantly aid the identification of IPG-separated proteins. However, the small size of the peptides leads to diffusion along and outside the gel matrix. In this study, we describe a simple spray-based procedure to perform 'on-strip' trypsin digestion of proteins embedded in IPG strips. Examination of intact myoglobin and its tryptic peptides shows that post-digestion diffusion of tryptic peptides is significantly minimized using this approach.