Rapid Protein Digestion Research Articles

Development of Rapid Analytical Methods in the Laboratory of Pharmaceutical Analytical Chemistry (LCAP)

The use of rapid and ultra-rapid analytical methods in the pharmaceutical domain is mandatory in the drug discovery, drug development, and quality control steps. Liquid chromatography (LC) and capillary electrophoresis (CE) are therefore particularly studied in the Laboratory of Pharmaceutical Analytical Chemistry (LCAP) to perform fast and ultra-fast analyses of drugs present in different matrices. Due to its sensitivity and selectivity, mass spectrometry (MS) is considered today as the gold standard in pharmaceutical analysis and has been generally used as the detector of choice for performing rapid analyses. This article gives a survey of the applied strategies achieved in the LCAP for the last five years such as: use of new chromatographic supports and small columns in LC; development of column switching techniques with extraction supports compatible with the direct injection of biological matrices; use of immobilized enzyme reactors for rapid protein digestion; enhancement of electrophoretic mobility and development of different injection procedures in CE.

Miniaturized membrane-based reversed-phase chromatography and enzyme reactor for protein digestion, peptide separation, and protein identification using electrospray ionization mass spectrometry.

A commonly used capillary fitting is employed for housing miniaturized membrane chromatography for performing reversed-phase peptide separations. By placing a hydrophobic and porous polyvinylidene fluoride membrane around the end of a polymer sleeve, the assembly of capillary fitting not only provides the stationary phase, but also establishes the necessary flow paths using capillary connections. The miniaturized membrane chromatography system is coupled with a micro-enzyme reactor containing immobilized trypsins for performing rapid protein digestion, peptide separation, and protein identification using electrospray ionization mass spectrometry. Separation performance of cytochrome c digest in miniaturized membrane chromatography is compared with the results obtained from micro-LC and capillary LC. The efficacy and the potentials of miniaturized membrane chromatography in tryptic mapping are reported. The use of miniaturized membrane chromatography allows significant reduction in sample consumption together with enhanced detection sensitivity. By minimizing the void volume in miniaturized membrane chromatography, the elution times of cytochrome c peptides are significantly shortened in this study in comparison with our previous results, and are comparable with those in micro-LC and capillary LC using considerably higher mobile phase flow-rates.

Microfluidic components for protein characterization

The use of microfluidic components to create an analytical toolbox for the very rapidly growing field of proteomics is described. This toolbox provides novel generic analytical solutions that are highly adaptable for analysis of various biomolecules, ranging from high to low abundant. The components are fabricated using silicon micromachining and consist of a microchip immobilised enzyme reactor (μIMER), a piezoelectric microdispenser and high-density nanovial target plates. This microtechnology based platform interfaces matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI TOF-MS) to a wide range of upstream sample handling and/or analytical techniques. Examples of applications such as rapid on-line digestion (12 s) and sample preparation of proteins, interfacing to capillary liquid chromatography (100 attomol sensitivity), and in-vial chemistry on femtomol amounts of sample are presented.

On-line coupling of micro-enzyme reactor with micro-membrane chromatography for protein digestion, peptide separation, and protein identification using electrospray ionization mass spectrometry

To miniaturize high-performance membrane chromatography, a poly(vinylidene fluoride) membrane medium, employed as the stationary phase, is sandwiched between two poly(dimethylsiloxane) substrates containing the microchannels. The microchannels are fabricated by the capillary molding technique, involving the use of capillaries as the channel template and the fluid inlet/outlet. The micro(μ)-membrane chromatography system is coupled with a μ-enzyme reactor containing immobilized trypsins for performing rapid protein digestion, peptide separation, and protein identification using electrospray ionization mass spectrometry. Separation performance of cytochrome c digest in μ-membrane chromatography is compared with the results obtained from a regular reversed-phase μ-liquid chromatography. The efficacy and the potentials of μ-membrane chromatography in tryptic mapping are reported. On-line integration of the μ-enzyme reactor with μ-chromatographic separation techniques and electrospray ionization mass spectrometry clearly provides a microanalytical platform for automated sample handling, minimized sample loss, and reduced sample consumption. It also provides enhanced detection sensitivity and dynamic range for the analysis of complex protein mixtures such as cell lysates in proteomics research.

On-chip proteolytic digestion and analysis using "wrong-way-round" electrospray time-of-flight mass spectrometry.

Rapid protein digestion and analysis using a hybrid microchip nanoelectrospray device and time-of-flight mass spectrometry detection are reported. The device consists of a planar glass chip with microfabricated channels coupled to a disposable nanospray emitter. Reactions between substrate and enzyme (trypsin), mixed off-chip and then immediately loaded into a sample reservoir on the device, are monitored in real time following the onset of electrospray. Protein cleavage products are determined at the optimum pH for generating tryptic fragments, directly from the digestion buffer using "wrong-way-round" electrospray, i.e., monitoring (MH)+ ions from basic solutions. Intense tryptic peptide ions are observed within a few minutes following sample loading on the microchip. Proteins were identified from low femtomole or even attomole quantities of analyte/spectrum using peptide mass fingerprinting, loading 0.1-2 pmol/microL of sample on the chip. The sequence coverage for analyzed proteins ranged from 70 to 95%. The rapid analysis of human hemoglobin is demonstrated using the technique.

Integration of immobilized trypsin bead beds for protein digestion within a microfluidic chip incorporating capillary electrophoresis separations and an electrospray mass spectrometry interface

A microfluidic device is described in which an electrospray interface to a mass spectrometer is integrated with a capillary electrophoresis channel, an injector and a protein digestion bed on a monolithic substrate. A large channel, 800 microm wide, 150 microm deep and 15 mm long, was created to act as a reactor bed for trypsin immobilized on 40-60 microm diameter beads. Separation was performed in channels etched 10 microm deep, 30 microm wide and about 45 mm long, feeding into a capillary, attached to the chip with a low dead volume coupling, that was 30 mm in length, with a 50 microm i.d. and 180 microm o.d. Sample was pumped through the reactor bed at flow rates between 0.5 and 60 microL/min. The application of this device for rapid digestion, separation and identification of proteins is demonstrated for melittin, cytochrome c and bovine serum albumin (BSA). The rate and efficiency of digestion was related to the flow rate of the substrate solution through the reactor bed. A flow rate of 1 or 0.5 microL/min was found adequate for complete consumption of cytochrome c or BSA, corresponding to a digestion time of 3-6 min at room temperature. Coverage of the amino acid sequence ranged from 92% for cytochrome c to 71% for BSA, with some missed cleavages observed. Melittin was consumed within 5 s. In contrast, a similar extent of digestion of melittin in a cuvet took 10-15 min. The kinetic limitations associated with the rapid digestion of low picomole levels of substrate were minimized using an integrated digestion bed with hydrodynamic flow to provide an increased ratio of trypsin to sample. This chip design thus provides a convenient platform for automated sample processing in proteomics applications.

Studies on the relationship between the degradative rates of proteins in vivo and their isoelectric points.

Acidic proteins tend to be degraded more rapidly than neutral or basic proteins in rat liver, skeletal muscle, kidney and brain and in mouse liver and skeletal muscle. We now report a similar relationship among soluble proteins from rat lung, heart and testes, and from human fibroblasts and mouse-embryo cells grown in culture. These findings indicate that the correlation between protein net charge and degradative rate is a general characteristic of intracellular protein degradation in mammals. This relationship between isoelectric point and half-life appears to be distinct from the previously reported correlation between subunit molecular weight and protein half-lives. The more rapid degradation of acidic proteins does not result from their being of larger molecular weight than neutral or basic proteins. Furthermore, proteins within specific isoelectric point ranges still exhibit a relationship between subunit size and half-life. Finally, a group of membrane or organelle-associated proteins that are insoluble in phosphate-buffered saline and water but soluble in 1% Triton X-100 exhibit a correlation between size and half-life, but not between net charge and half-life. The biochemical reasons for the relationship between protein isoelectric point and half-life are unclear, although several possible explanations are presented. It is not due to a greater sensitivity of acidic proteins to proteolytic attack since experiments with a variety of endoproteinases, including trypsin, chymotrypsin, Pronase, papain, chymopapain, Staphylococcus aureus V8 proteinase, pepsin and lysosomal cathepsins from rat liver, have failed to demonstrate more rapid digestion of acidic proteins.

Rapid protein uptake and digestion in proximal tubule lysosomes

The aim of the present study was to determine the initial time course for the handling of protein by the proximal tubule cells in rat kidney, using 125I-labeled cytochrome C as a tracer. The uptake of the protein was followed by electron microscope autoradiography in kidneys from rats fixed by perfusion after i.v. injection of cytochrome C. Protein degradation was studied by incubating cortical slices, taken from rats, injected with the same label, and measuring the release of trichloroacetic acid (TCA)-soluble radioactivity from the slices. It was shown by autoradiography that lysosomes in proximal tubule cells start to accumulate labeled cytochrome C within 3 min after injection of the protein, and that the concentration of label in lysosomes increases during the first 30 min after injection, whereas it decreases in endocytic vacuoles. The catabolism of protein as measured in cortical slices began within 13 min after the i.v. injection. Other experiments showed that the accumulation of cytochrome C in the kidneys is very fast. The maximum accumulation, 37% of the injected dose, was reached seven minutes after injection. The results show that protein uptake in proximal tubule cells, transport into lysosomes and the digestion of protein is a more rapid process than previously reported in ultrastructural studies.

Rumen Digestion Studies: II. Studies in the Chemistry of Rumen Digestion

Studies of the chemical changes in the rumen suggest the following salient features regarding the fate of roughage in the ruminant digestive tract: During the first 6 hours after feeding the predominant phenomenon was the rapid digestion of proteins and carbohydrates. Decomposition of cellulose began during this period but was not extensive. The predominant phenomenon during the second 6 hours was the rapid disintegration of cellulose. Digestion of proteins and carbohydrates in this period paralleled the digestion of cellulose. Although small amounts of lignin may be attacked in the rumen, functionally the rumen is not a factor in lignin digestion. Rumen digestion was observed to come to a standstill within 12 hours after feeding. Prolonging the digestion periods to 24 hours did not increase rumen digestion coefficients. Lignin apparently protects plant fragments from further action by rumen microflora. Average rumen digestion coefficients for 8 trials at 12–14 hours after feeding were: dry matter 48.4, protein 59.6, nitrogen-free extract 65.2, crude fiber 27.2, cellulose 43.4, other carbohydrates 83.0 and lignin 3.1%. After plant fragments pass from the rumen variable amounts of lignin may be digested, thereby exposing varying amounts of cellulose and protein to further digestion. Fecal digestion was observed to be quite variable even though rumen digestion of the same lots of hay was very constant. The caecum plays only a supplementary role in the disintegration of roughage within the ruminant organism. An average of 11.6% of the cellulose and possibly 9.5% protein was observed to be digested in the caecum. The production of fatty acids in the rumen was demonstrated. Maximum increases, after the increase due to digestion was eliminated, were 54.9% on an alfalfa hay ration and 281.7% on a beet pulp ration. The rapid dissipation of fatty acids from the rumen together with the marked increases noted suggests that fatty acids may make a highly significant contribution to the nutrition of the ruminant either as an intermediate or end product of digestion.