原子转移自由基聚合修饰银丝固定化酶反应器的制备及在蛋白质组鉴定中的应用

针对传统溶液酶解存在的酶解时间较长、酶自切物干扰以及蛋白酶不能重复使用等缺陷,通过电子转移生成催化剂的原子转移自由基聚合法修饰银丝,并以其为载体制备了一种新型的固定化酶反应器。用质谱考察了银丝固定化酶反应器(SW-Trypsin)的酶解效率、重复性和回收率。结果表明:绒毛状聚合物修饰的SW-Trypsin的酶解效率较高,酶解标准蛋白牛血清白蛋白(BSA)20 min后,肽段的氨基酸序列覆盖率可达93%,高于传统溶液酶解方法酶解16 h所得79%的覆盖率。使用该固定化酶反应器于一个月内8次酶解BSA所得的氨基酸序列覆盖率在89%到97%之间,平均覆盖率为94%,显示出良好的稳定性。另外,该固定化酶反应器酶解马心肌红蛋白(MYO)的回收率为87.67%。最后,用SW-Trypsin酶解腾冲嗜热菌全蛋白20 min,所鉴定到的氨基酸序列覆盖率和蛋白数量与同样条件下溶液酶解16 h的结果接近,且零漏切位点肽段的比例更高。加之容易分离的优点,SW-Trypsin在蛋白质组学的应用中具有良好的前景。     

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.     

Accelerated 18O-labeling in urinary proteomics

Proteolytic (18)O-labeling of peptides has been studied and optimized in order to improve the labeling efficiency and to accelerate the process without increasing the degree of incomplete labeling. Using peptides generated from tryptic digested bovine serum albumin (BSA) and cytochrome c as model proteins, it was shown that complete labeling was achieved after 2 h at pH 6. To increase the sample throughput in a bottom-up proteomic setup, tryptic digestion of proteins in-solution was replaced with tryptic digestion using immobilized trypsin. As a result, an integrated approach was made possible, where both digestion (pH 8) and (18)O/(16)O-labeling of the resulting peptides (pH 6) were done using immobilized trypsin beads. This simplified the sample handling and reduced the overall reaction time significantly: the setup enabled tryptic digestion and (18)O/(16)O-labeling without sample transfer steps within 3.5 h with average (18)O/(16)O-ratios of 0.96±0.13 in aqueous buffer. The initial results were confirmed with a more complex matrix, by spiking urine with the model proteins, yielding results comparable with the ratios obtained in buffer. Satisfying ratios were also achieved regarding urinary proteins identified in a full scale bottom-up experiment. Average (18)O/(16)O-peptide ratios of 0.83±0.13 and 0.91±0.27 indicated good performance in a highly relevant matrix for biomarker discovery.     

Characterization of a monolithic immobilized trypsin microreactor with on-line coupling to ESI-MS

The preparation and characterization of a miniaturized trypsin reactor using on-line coupling with an ESI-TOF mass spectrometer are described. L-1-Tosylamido-2-phenylethyl chloromethyl ketone-trypsin was covalently immobilized on poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith prepared in a 75 microm ID fused silica capillary resulting in a bioreactor with high local concentration of the proteolytic enzyme. Covalent immobilization of trypsin on this support was performed using the epoxide functional groups in either a one- or a multistep reaction. For on-line protein digestion-MS analysis the bioreactor was coupled with the mass spectrometer using a liquid junction microelectrospray interface. The performance of the reactor was tested using an on-line flow through the system with flow rates of 50-300 nL/min. The resulting protein consumption was in the atto- to low femtomole range. Proteolytic activity was characterized in a wide range of conditions with respect to the flow rate, pH, and temperature. Complete protein digestion was achieved in less than 30 s at 25 degrees C with the sequence coverage of 80% (cytochrome c), which is comparable to 3 h digestion in solution at 37 degrees C. Besides the good performance at laboratory temperature, the immobilized trypsin in the bioreactor also performed well at lower pH compared to the standard in-solution protocols.     

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.     

Highly efficient enrichment and subsequent digestion of proteins in the mesoporous molecular sieve silicate SBA-15 for matrix-assisted laser desorption/ionization mass spectrometry with time-of-flight/time-of-flight analyzer peptide mapping

Based on a previous study of protein digestion inside the nanoreactor channels of the mesoporous molecular sieve silicate SBA-15 (Chem. Eur. J. 2005, 11: 5391), we have developed a highly efficient enrichment and subsequent tryptic digestion of proteins in SBA-15 for matrix-assisted laser desorption/ionization mass spectrometry with time-of-flight/time-of-flight analyzer (MALDI-TOF/TOF) peptide mapping. The performance of the method is exemplified with myoglobin and cytochrome c. First, protein adsorption isotherms for two standard proteins with a range of initial concentration of proteins were investigated at room temperature. The results revealed that the kinetic adsorption rate of a protein within SBA-15 was independent of initial protein concentration, and a 15-min protein enrichment within SBA-15 could be enough for protein identification in biological samples. It was noticed that no washing steps were needed to avoid protein loss due to desorption from the mesochannels into solution. Second, protein digestion inside the channels of SBA-15 was also optimized. After adsorption of proteins into SBA-15 in 15 min, the trypsin solution (pH 8) was directly added to the SBA-15 beads with immobilized proteins by centrifugation, and then the digestion was performed for 15 min at 37 degrees C. It was observed that a higher peptide sequence covering of 98% for myoglobin was obtained by MALDI-TOF/TOF analysis, compared to in-solution digestion. So the protein digestion inside SBA-15 was proved to be significantly faster and yielded a better sequence coverage. The new procedure allows for rapid protein enrichment and digestion inside SBA-15, and has great potential for protein analysis.     

新型亲水聚合物修饰硅胶颗粒固定化酶的制备及在蛋白质组鉴定中的应用

采用原子转移自由基聚合法制备了亲水聚合物修饰的硅胶颗粒作为一种新型固定化酶载体,在实现胰蛋白酶高密度固定的同时,显著降低了载体材料非特异性吸附导致的样品损失。因此,该固定化酶材料兼具高酶解效率和高回收率的特性。以标准蛋白质牛血清白蛋白(BSA)为样本,使用该固定化酶1 min即可完成酶解,鉴定到肽段对BSA的氨基酸序列覆盖率可达90%以上。该固定化酶材料成功应用于酵母菌全蛋白质复杂样本的酶解,从3 min酶解产物中鉴定到666个蛋白质,超过同样条件下溶液酶解12 h的鉴定结果。     

Rapid and Efficient Proteolysis for Protein Analysis by an Aptamer-Based Immobilized Chymotrypsin Microreactor

Efficient protein digestion is a key step for successful mass spectrometry identification. However, traditional in-solution digestion suffers some drawbacks, such as autolysis of protease, long analysis times and lack of control. Recently, specific single-stranded nucleic acids, aptamers, screened from random sequence pools, have been performed high affinity for targets. In this paper, we have developed a novel enzyme reactor, which immobilized chymotrypsin based on aptamer-grafted silica beads. Mixed proteins, which consist of bovine serum albumin, myoglobin, and cytochrome c, were used as samples, to evaluate the digestion performance of the enzymatic reactor. With the use of this novel tool, proteins were digested in 40 min to an extent similar to that achieved with soluble enzyme at 37°C after 16 h. Moreover, enzymatic reactor regeneration was carried out through chymotrypsin elution and re-immobilization. The advanced characteristics of the aptamer-based chymotrypsin reactor demonstrated that aptamers could serve as novel materials for rapid and efficient enzyme immobilization and application in protein studies.     

Immobilization of enzyme on detonation nanodiamond for highly efficient proteolysis

Immobilization of enzyme on detonation nanodiamond (dND, 3-10 nm) and its application for efficient proteolysis have been demonstrated. By evaluation of the Michaelis constant (K_m) and maximum velocity (V_max) of immobilized enzyme, its activity was not impaired significantly by immobilization. And enzyme immobilized on dNDs exhibited much better thermal and chemical stabilities than its free counterpart and maintained high activity even after 10 times reuse. The efficient proteolysis by trypsin immobilized on dNDs (dND-trypsin) is demonstrated with the digestion of myoglobin (or other model protein) in a short time (5 min). Large numbers of identified peptides obtained by dNDs-trypsin enable a higher degree of sequence coverage and more positive identification of proteins than those obtained by in-solution digestion and the commercial immobilized trypsin beads, respectively. Moreover, immobilization of peptide-N-glycosidase F (PNGase F) on dNDs was realized and resulted in faster sequential glycosidase digestion of glycopeptides in less than 10 min.     

Ion-exchange-membrane-based enzyme micro-reactor coupled online with liquid chromatography-mass spectrometry for protein analysis

In this article, we developed a membrane-based enzyme micro-reactor by directly using commercial polystyrene–divinylbenzene cation–exchange membrane as the support for trypsin immobilization via electrostatic and hydrophobic interactions and successfully applied it for protein digestion. The construction of the reactor can be simply achieved by continuously pumping trypsin solution through the reactor for only 2 min, which was much faster than the other enzyme immobilization methods. In addition, the membrane reactor could be rapidly regenerated within 35 min, resulting in a “new” reactor for the digestion of every protein sample, completely eliminating the cross-interference of different protein samples. The amount and the activity of immobilized trypsin were measured, and the repeatability of the reactor was tested, with an RSD of 3.2% for the sequence coverage of cytochrome c in ten digestion replicates. An integrated platform for protein analysis, including online protein digestion and peptide separation and detection, was established by coupling the membrane enzyme reactor with liquid chromatography–quadrupole time-of-flight mass spectrometry. The performance of the platform was evaluated using cytochrome c, myoglobin, and bovine serum albumin, showing that even in the short digestion time of several seconds the obtained sequence coverages was comparable to or higher than that with in-solution digestion. The system was also successfully used for the analysis of proteins from yeast cell lysate.     

基于温敏型材料的固定化酶的制备及其在蛋白质组快速分析中的应用

蛋白质组学通过规模化鉴定、分析从细胞、组织或有机体中提取的蛋白质,从而获得蛋白表达、修饰、组成和定量的变化信息。在目前最为有效的“鸟枪法”蛋白质组学策略中,固定化酶试剂基质常用固相载体材料,该固定化酶试剂在酶解蛋白质时为异相体系,存在固液界面传质阻力和空间位阻,限制了酶解效率和样品处理通量。针对这一技术瓶颈,本研究利用温敏聚合物对外界温度变化的响应能力,制备了一种新型的基于可溶性温敏聚合物的固定化胰蛋白酶试剂。该固定化酶特有的温度敏感特性,使其具有“高温均相酶解,低温异相分离”的特色,且兼具酶切时间显著缩短、酶可重复利用的优势。 BSA 1 min固定化酶解产物肽段的氨基酸序列覆盖率可达94%,高于传统溶液酶解12 h所得覆盖率为(74%)。进一步将该固定化酶试剂应用于HeLa细胞全蛋白质组的酶解,其酶解效果与相同条件下溶液酶解12 h相当。该固定化酶试剂对复杂蛋白质的快速、高效酶解充分证明其在蛋白质组学研究中的应用潜力。     

固定化胰蛋白酶整体小柱的制备及其用于微量蛋白质的快速酶解

发展了一种光引发聚合法制备固定化胰蛋白酶整体小柱的方法,以用于微量蛋白质的快速酶解。整体小柱由功能单体4-戊烯酸琥珀酰亚胺酯、甲基丙烯酸羟乙酯,交联剂季戊四醇三丙烯酸酯和三元致孔剂二甲基亚砜、N,N-二甲基甲酰胺、十二醇在20 μL的移液器吸头尖端原位聚合而成。形成整体柱后,胰蛋白酶分子通过氨基与琥珀酰亚胺酯反应实现固定化。系统研究了聚合溶液中活性酯含量与柱床体积对胰蛋白酶固载量的影响,评价了固定化酶整体小柱对标准蛋白细胞色素C和牛血清白蛋白的酶解效率,以及整体小柱的稳定性和重复性。结果表明,在离心辅助下,酶解过程可在10 min内完成,批次间具有良好的重复性。最后将固定化酶整体小柱应用于1×10⁵个人急性早幼粒白血病（NB4）细胞与人急性T细胞白血病（Jurkat T）细胞的快速酶解,经纳升级液相色谱与高分辨质谱联用分析后鉴定得到2489个和2572个蛋白质。相比于溶液状态下的酶解,分别提高了2.2%和6.1%的蛋白鉴定数量,展现了其在蛋白组学研究中的应用潜力。     

Rapid and efficient proteolysis through laser-assisted immobilized enzyme reactors

In this report, laser radiation (808nm) for the first time was employed to enhance the efficiency of proteolysis through immobilized enzyme reactor (IMER). IMER based monolithic support was prepared in the fused-silica capillary via a simple two-step procedure including acryloylation on trypsin surface and in situ aqueous polymerization/immobilization. The feasibility and high efficiency of the laser-assisted IMER were demonstrated by the digestion of bovine serum albumin (BSA), cytochrome c (Cyt-c) and β-casein. The digestion process was achieved in 60s. The peptides were identified by MALDI-TOF-MS, yielding the sequence coverage of 33% for BSA, 73% for Cyt-c and 22% for β-casein. The comparisons between the in-solution digestion and on IMER reaction with/without laser assistance were made. To further confirm its efficiency in proteome analysis, the laser-assisted IMER was also applied to the analysis of one fraction of human serum sample through two-dimensional (2-D) separation of strong anion exchange/reversed-phase liquid chromatography (SAX/RPLC). After a database search, 49 unique peptides corresponding to 5 proteins were identified. The results showed that the laser-assisted IMER provides a promising platform for the high-throughput protein identification.     

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.     

High throughput tryptic digestion via poly (acrylamide-co-methylenebisacrylamide) monolith based immobilized enzyme reactor

A poly (acrylamide-co-methylenebisacrylamide) (poly (AAm-co-MBA)) monolith was prepared by thermal polymerization in the 100 or 250 μm i.d. capillary. The monolithic support was activated by ethylenediamine followed by glutaraldehyde. Trypsin was then introduced to form an immobilized enzyme reactor (IMER). The prepared IMER showed a reliable mechanical stability and permeability (permeability constant K = 2.65 × 10⁻¹³ m²). With BSA as the model protein, efficient digestion was completed within 20 s, yielding the sequence coverage of 57%, better than that obtained from the traditional in-solution digestion (42%), which took about 12 h. Moreover, BSA down to femtomole was efficiently digested by the IMER and positively identified by matrix assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). To test the applicability of IMER for complex sample profiling, proteins extracted from Escherichia coli were digested by the IMER and further analyzed by nanoreversed phase liquid chromatography-electrospray ionization-mass spectrometry (nanoRPLC-ESI-MS/MS). In comparison to in-solution digestion, despite slightly fewer proteins were positively identified at a false discovery rate (FDR) of ∼1% (333 vs 411), the digestion time used was largely shortened (20 s vs 24 h), implying superior digestion performance for the high throughput analysis of complex samples.     

A nanoporous reactor for efficient proteolysis

A nanoreactor based on mesoporous silicates is described for efficient tryptic digestion of proteins within the mesochannels. Cyano-functionalized mesoporous silicate (CNS), with an average pore diameter of 18 nm, is a good support for trypsin, with rapid in situ digestion of the model proteins, cytochrome c and myoglobin. The generated peptides were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Proteolysis by trypsin-CNS is much more efficient than in-solution digestion, which can be attributed to nanoscopic confinement and concentration enrichment of the substrate within the mesopores. Proteins at concentrations of 2 ng muL(-1) were successfully identified after digestion for 20 min. A biological complex sample extracted from the cytoplasm of human liver tissue was digested by using the CNS-based reactor. Coupled with reverse-phase HPLC and MALDI-TOF MS/MS, 165 proteins were identified after standard protein data searching. This nanoreactor combines the advantages of short digestion time with retention of enzymatic activity, providing a promising way to advance the development of proteomics.     

Preparing a metal-ion chelated immobilized enzyme reactor based on the polyacrylamide monolith grafted with polyethylenimine for a facile regeneration and high throughput tryptic digestion in proteomics

Initially, a poly (glycidyl methacrylate-co-acrylamide-co-methylenebisacrylamide) monolith was prepared in the 100 μm i.d. capillary, and then was grafted with polyethylenimine (Mw, ∼25,000) for adsorbing Cu²⁺, followed by chelating trypsin. As a result, efficient digestion for BSA (100 ng/μL) was completed within 50 s via such immobilized enzyme reactor (IMER); yielding 47% sequence coverage by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis. Compared with the conventional method for preparing the metal-ion chelated IMER, the regeneration of such IMER can be achieved facilely by the respective 30 min desorption and re-adsorption of trypsin, and 51% sequence coverage was obtained for 50 s BSA digestion after regeneration. BSA down to femtomole was also efficiently digested by the prepared regenerable IMER. Meanwhile, after the consecutive digestion of myoglobin and BSA, there was not any mutual interference for both during MALDI-TOF MS identification, indicating the low nonspecific adsorption of such regenerable IMER. To test the applicability of regenerable IMER for complex sample profiling, proteins (150 ng) extracted from Escherichia coli were digested within 80 s by the regenerable IMER and further analyzed by nanoreversed phase liquid chromatography–electrospray ionization–mass spectrometry successfully, showing its practicability for the high throughput analysis of complex samples.     

Efficient Proteolysis of Glycoprotein Using a Hydrophilic Immobilized Enzyme Reactor Coupled with MALDI-QIT-TOF-MS Detection and μHPLC Analysis

A hydrophilic immobilized enzyme reactor (IMER) containing trypsin was prepared and applied in the proteolysis of glycoproteins. Glycoproteins including horseradish peroxidase, asialofetuin, and fetuin were used to evaluate the performance of the hydrophilic IMER for the glycoprotein digestion. The digested products were detected by matrix-assisted laser desorption/ionization quadruple ion trap time-of-flight mass spectrometry and micro-high-performance liquid chromatography. The hydrophilic IMER showed higher enzymatic digestion efficiency compared with conventional in-solution digestion. The digestion time could be reduced from 16 h to several minutes. Furthermore, using microwaves as a heat source, the reproducibility of the hydrophilic IMER was evaluated and this IMER could be recycled for at least ten times without obvious loss of enzyme activity. The hydrophilic IMER provides a promising tool for high-throughput glycoproteome analysis.     

On-column digestion of proteins in aqueous-organic solvents

Proteolytic digestion is an important step in protein identification by peptide mass mapping and tandem mass spectrometry (MS/MS)-based peptide sequencing. Traditional methods of protein digestion require extended incubation times and have difficulty with proteolytically resistant proteins. Here, we describe a method in which a protein solution was combined with a mixed aqueous-organic solution (methanol, isopropanol, or acetonitrile) and passed through a microcolumn containing immobilized trypsin. Myoglobin sequence coverage was high (>85%) in all three solvents, and differences in spectra were seen among the different solution conditions. Notably, methanol-based digestions produced fewer missed cleavages while acetonitrile-based digestions produced the most peptides and the most intense mass spectra. Flow rates through the column were varied from 0.5 to 15 micro L/min, corresponding to column residence times of 78 and 2.6 s, respectively. All flow rates produced high sequence coverage of myoglobin, although, at higher flow rates, more missed cleavages were observed. No significant increase in undigested myoglobin was observed with flow rates up to 15 micro L/min. The described method was applied to the digestion of human transferrin (hTf), a proteolytically resistant protein. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometric (MALDI-TOFMS) analysis detected 42 peptides covering 46% of the hTf sequence. The traditional aqueous method resulted in 12 peptides (8% sequence coverage) only when high concentrations of trypsin were used. Lastly, digestion of low nanomolar myoglobin was shown to produce detectable peptides and resulted in a correct database hit. Thus, we demonstrate a method that is capable of rapid on-line digestion, thereby lending itself to high-throughput identification of proteins.     

A replaceable microreactor for on-line protein digestion in a two-dimensional capillary electrophoresis system with tandem mass spectrometry detection

We describe a two-dimensional capillary electrophoresis system that incorporates a replaceable enzymatic microreactor for on-line protein digestion. In this system, trypsin is immobilized on magnetic beads. At the start of each experiment, old beads are flushed to waste and replaced with a fresh plug of beads, which is captured by a pair of magnets at the distal tip of the first capillary. For analysis, proteins are separated in the first capillary. A fraction is then parked in the reactor to create peptides. Digested peptides are periodically transferred to the second capillary for separation; a fresh protein fraction is simultaneously moved to the reactor for digestion. An electrospray interface is used to introduce peptides into a mass spectrometer for analysis. This procedure is repeated for several dozen fractions under computer control. The system was demonstrated by the separation and digestion of insulin chain b oxidized and β-casein as model proteins.