MALDI-target integrated platform for affinity-captured protein digestion

To address immunocapture of proteins in large cohorts of clinical samples high throughput sample processing is required. Here a method using the proteomic sample platform, ISET (integrated selective enrichment target) that integrates highly specific immunoaffinity capture of protein biomarker, digestion and sample cleanup with a direct interface to mass spectrometry is presented. The robustness of the on-ISET protein digestion protocol was validated by MALDI MS analysis of model proteins, ranging from 40 fmol to 1 pmol per nanovial. On-ISET digestion and MALDI MS/MS analysis of immunoaffinity captured disease-associated biomarker PSA (prostate specific antigen) from human seminal plasma are presented.     

Disposable polymeric high-density nanovial arrays for matrix assisted laser desorption/ionization-time of flight-mass spectrometry: I. Microstructure development and manufacturing.

In order to meet the expected enormous demand for mass spectrometry (MS) throughput as a result of the current efforts to completely map the human proteome, this paper presents a new concept for low-cost high-throughput protein identification by matrix assisted laser desorption/ionization-time of flight-(MALDI-TOF)-MS peptide mapping using disposable polymeric high-density nanovial MALDI target plates. By means of microfabrication technology precision engineered nanovial arrays are fabricated in polymer substrates such as polymethylmethacrylate (PMMA) and polycarbonate (PC). The target plate fabrication processes investigated were precision micromilling, cold embossing and injection moulding (work in progress). Nanovial dimensions were 300, 400 or 500 microm. Typical array densities were 165 nanovials/cm2, which corresponds to 3,300 vials on a full Applied Biosystems MALDI target plate. Obtained MALDI data displayed equal mass resolution, accuracy, signal intensity for peptide standards as compared to high-density silicon nanovial arrays previously reported by our group [7], as well as conventional stainless steel or gold targets.     

Characterization of femtomole levels of proteins in solution using rapid proteolysis and nanoelectrospray ionization mass spectrometry

A method for the rapid proteolytic digestion of low picomole to low femtomole amounts of proteins in solution using a capillary immobilized protease column is presented. Dilute protein samples are passed through a “μ-digestion” column packed with Poroszyme^? immobilized trypsin for generation of proteolytic fragments in less than 10 min. After digestion, nanoelectrospray ionization mass spectrometry (NanoES) is used to generate a peptide map, and peptides of interest are subjected to MS/MS from the same sample. By digesting only 100 fmol of the protein src kinase and 30 fmol of the protein lck kinase with a tryptic μ-digestion column, we obtained sufficient data from NanoES-MS and MS/MS to unambiguously identify both proteins using database searching. This approach was also used to locate a phosphorylation site on lck kinase starting with only 300 fmol of protein. The method was successfully used to identify an E. coli cold shock protein in a fraction collected from a two-dimensional HPLC separation of an E. coli cell lysate. The μ-digestion column was found to be less susceptible to adsorptive losses than solution digests, thus allowing for digestion and enhanced recovery of peptides from even low fmol amounts of protein in solution.     

Development of an automated digestion and droplet deposition microfluidic chip for MALDI-TOF MS

An automated proteolytic digestion bioreactor and droplet deposition system was constructed with a plastic microfluidic device for off-line interfacing to matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The microfluidic chips were fabricated in poly(methyl methacrylate) (PMMA), using a micromilling machine and incorporated a bioreactor, which was 100 microm wide, 100 microm deep, and possessed a 4 cm effective channel length (400 nL volume). The chip was operated by pressure-driven flow and mounted on a robotic fraction collector system. The PMMA bioreactor contained surface immobilized trypsin, which was covalently attached to the UV-modified PMMA surface using coupling reagents N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) and hydroxysulfosuccinimide (sulfo-NHS). The digested peptides were mixed with a MALDI matrix on-chip and deposited as discrete spots on MALDI targets. The bioreactor provided efficient digestion of a test protein, cytochrome c, at a flow rate of 1 microL/min, producing a reaction time of approximately 24 s to give adequate sequence coverage for protein identification. Other proteins were also evaluated using this solid-phase bioreactor. The efficiency of digestion was evaluated by monitoring the sequence coverage, which was 64%, 35%, 58%, and 47% for cytochrome c, bovine serum albumin (BSA), myoglobin, and phosphorylase b, respectively.     

Capillary high-performance liquid chromatography/electrospray ion trap time-of-flight mass spectrometry using a novel nanoflow gradient generator

A type of high-performance liquid chromatography (HPLC) based on a novel nanoflow gradient generator (Asymptotic-Trace-10-Port-Valve (AT10PV) nanoGR generator) was developed and coupled with an electrospray ion trap time-of-flight mass spectrometer (ESI-IT-TOF MS). Stability of the nanoflow GR HPLC system was tested at flow rates of 20 and 50 nL/min by using a nanoflow meter. Average flow rates in a 2-h run were 51.2 nL/min with RSD 0.7% and 21.0 nL/min with RSD 1.8%. Repeatability of analysis of the nanoHPLC/ESI-IT-TOF MS system was also tested by injecting 1.0 microL of trypsin digested bovine serum albumin (BSA) (100 fmol) into a monolithic silica-ODS column (30 microm i.d., 150 mm in length) through a packed silica-ODS trapping column (particle size 5 microm, 150 microm i.d., 10 mm in length). At a flow rate of 50 nL/min, the result demonstrated a reasonably good repeatability of peak retention times (RSD: 0.32-1.1%) and base-ion peak areas (RSD: 4.4-6.6%).     

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.     

小分子表面活性剂在胶内酶切增效中的应用

本研究成功地将一种有机小分子表面活性剂RapiGest SF(Waters)用于改进电泳分离的蛋白质的鉴定效率。通过基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)的肽质量指纹谱,考察了酶切时间、加入量、加样次序、点靶方法对方法灵敏度、蛋白质鉴定率的影响。RapiGest SF浓度为0.5%~1%,在酶切之前加入可获得更多的肽段峰和更高的鉴定率。本方法考染体系的总灵敏度为332fmol,银染体系为664fmol。比较了RapiGest SF与MALDI-TOF-MS和电喷雾质谱(ESI-MS)兼容性,未观察到明显的负影响。方法操作简便,重复性较好,适合鉴定电泳分离的低丰度蛋白质。     

Rapid protein identification using a microscale electrospray LC/MS system on an ion trap mass spectrometer

A methodology has been developed for the rapid identification of gel separated proteins. Following in gel protein digestion with trypsin, the resulting peptide mixture is analyzed by on-line liquid chromatography, electrospray mass spectrometry (LC/MS). The mass spectral data containing either accurate mass values or sequence specific fragment ion information is then matched to a database of known protein sequences. Key features of the LC/MS system are the use of a novel integrated, microscale LC column-electrospray interface and variable flow solvent delivery to optimize the efficiency of sample loading and gradient elution. With these enhancements, only 10 min is required to analyze each sample. The method is routine for sample amounts ranging from 50 to 500 fmol. The analysis parameters for the ion trap mass spectrometer have to be carefully adjusted in order to keep pace with the rapidly eluting LC peaks. Although designed for rapid LC separations, the integrated column-electrospray interface is also able to provide extended analyses of selected components using a technique known as “peak parking. ”     

The resistance of metallothionein to proteolytic digestion: an LC-MS/MS analysis

Metallothioneins (MTs) are a family of cysteine-rich metalloproteins which strongly bind to heavy metals, such as Cd(II), Zn(II), and Cu(I). Previous works by other group using gel electrophoresis and fluorescence showed MTs were resistant to proteolytic digestion by a variety of enzymes, raising the difficulties in proteomic identification of MTs. The present work was attempted to analyze the resistance of MTs to trypsin using LC with MS/MS (LC-MS/MS), which was able to determine the sequences of the produced peptides and thus precisely characterize the cleavages. The results showed that metal-saturated MTs were completely resistant to trypsin. This resistance problem could be overcome by the addition of EDTA to MT samples, which rendered MTs readily digested into peptides and identified by MS/MS. Interestingly, the partially metal binding MTs were digested into peptides predominantly with miss cleavages which were well dependent on the amount of heavy metals bound to MTs. An explanation for these observations was proposed. The potential applications of the MT's resistance to trypsin in isolation and identification of MTs in complex mixtures such as cultured cells was demonstrated. The preliminary data also showed the same proteomic approach of proteolytic digestion followed by MS/MS analysis may provide information on metal binding status of MTs, along with the identification of MTs in a mixture.     

Disposable polymeric high-density nanovial arrays for matrix assisted laser desorption/ionization-time of flight-mass spectrometry: II. Biological applications.

A novel disposable high-density matrix assisted laser desorption/ionization (MALDI) target plate made either from polymethylmethacrylate (PMMA) or polycarbonate (PC) is presented where thousands (1,200-1,600) of samples can be deposited and subsequently analyzed by MALDI-time of flight (TOF) mass spectrometry. Good reproducibility was obtained across the plate regardless of position on the target plate with a relative standard deviation (RSD) on the peak intensity of typically 30% calculated from data generated by analysis of a 10 nm peptide mixture of angiotensin I, II, III and bradykinin. The nanovial array format combined with microdispensing technology makes it possible to carry out in-vial chemistry on deposited samples. This is demonstrated by the analysis of peptides from beta-casein and subsequent in-vial dephosphorylation of its phosphopeptides at 10 fmol levels by microdispensing of alkaline phosphatase, into the nanovial. The mass spectra obtained from these polymeric targets provides can also be used in high sensitivity applications as shown by peptide mass fingerprinting of human fibroblast proteins separated by two-dimensional gel electrophoresis.     

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.     

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。     

Ultratrace liquid chromatography/mass spectrometry analysis of large peptides with post-translational modifications using narrow-bore poly(styrene-divinylbenzene) monolithic columns and extended range proteomic analysis

This paper describes approaches to optimize the chromatographic performance for our recently developed LC-MS platform, extended range proteomic analysis (ERPA), for comprehensive protein characterization at the ultratrace level. Large digested peptide fragments up to 10 kDa (e.g., from lysyl endopeptidase digestion) with or without modifications were well separated with high resolution using narrow bore (20 and 50 microm I.D.) poly(styrene-divinylbenzene) (PS-DVB) monolithic columns constructed by in situ solution polymerization. Importantly, the macroporous structure of the monolithic columns facilitated mass transport of large peptides with improved recovery relative to small pore size reversed-phase packings. High sequence coverage (>95%), including identification of phosphorylated and glycosylated particles was achieved for beta-casein and epidermal growth factor receptor (EGFR) at the 4 and 20 fmol levels per injection, respectively, using the 20 microm I.D. PS-DVB monolithic column. For peptides with greater ionization efficiency, the detection limit could be lowered to approximately 400 zmol. Typically, the separation system produced a peak capacity of approximately 200 for a 10 cm column. This paper demonstrates that narrow-bore monolithic columns are suitable for high sensitivity and high-resolution separation of large peptide fragments by LC-MS analysis.     

Improving off-line accelerated tryptic digestion. Towards fast-lane proteolysis of complex biological samples

Off-line digestion of proteins using immobilized trypsin beads is studied with respect to the format of the digestion reactor, the digestion conditions, the comparison with in-solution digestion and its use in complex biological samples. The use of the filter vial as the most appropriate digestion reactor enables simple, efficient and easy-to-handle off-line digestion of the proteins on trypsin beads. It was shown that complex proteins like bovine serum albumin (BSA) need much longer time (89 min) and elevated temperature (37 degrees C) to be digested to an acceptable level compared to smaller proteins like cytochrome c (5 min, room temperature). Comparing the BSA digestion using immobilized trypsin beads with conventional in-solution digestion (overnight at 37 degrees C), it was shown that comparable results were obtained with respect to sequence coverage (>90%) and amount of missed cleavages (in both cases around 20 peptides with 1 or 2 missed cleavages were detected). However, the digestion using immobilized trypsin beads was considerable less time consuming. Good reproducibility and signal intensities were obtained for the digestion products of BSA in a complex urine sample. In addition to this, peptide products of proteins typically present in urine were identified.     

Gel-free sample preparation for the nanoscale LC-MS/MS analysis and identification of low-nanogram protein samples

Protein identification at the low nanogram level could in principle be obtained by most nanoscale LC-MS/MS systems. Nevertheless, the complex sample preparation procedures generally required in biological applications, and the consequent high risk of sample losses, very often hamper practical achievement of such low levels. In fact, the minimal amount of protein required for the identification from a gel band or spot, in general, largely exceeds the theoretical limit of identification reachable by nanoscale LC-MS/MS systems. A method for the identification of low levels of purified proteins, allowing limits of identification down to 1 ng when using standard bore, 75 microm id nanoscale LC-MS/MS systems is here reported. The method comprises an offline two-step sample cleanup, subsequent to protein digestion, which is designed to minimize sample losses, allows high flexibility in the choice of digestion conditions and delivers a highly purified peptide mixture even from "real world" digestion conditions, thus allowing the subsequent nanoscale LC-MS/MS analysis to be performed in automated, unattended operation for long series. The method can be applied to the characterization of low levels of affinity purified proteins.     

Disparities between immobilized enzyme and solution based digestion of transferrin with trypsin

Trypsin digestion is a major component of preparing proteins for peptide based identification and quantification by mass spectral (MS) analysis. Surprisingly proteolysis is the slowest part of the proteomics process by an order of magnitude. Numerous recent efforts to reduce protein digestion to a few minutes have centered on the use of an immobilized enzyme reactor (IMER) to minimize both trypsin autolysis and vastly increase the trypsin to protein ratio. A central question in this approach is whether proteolysis with an IMER produces the same peptide cleavage products as derived from solution based digestion. The studies reported here examined this question with transferrin; a model protein of known resistance to trypsin digestion. Results from these studies confirmed that a trypsin‐IMER can in fact digest transferrin in a few minutes; providing tryptic peptides that subsequent to MS analysis allow sequence identification equivalent to solution digestion. Although many of the peptides obtained from these two trypsin digestion systems were identical, many were not. The greatest difference was that the trypsin‐ IMER produces (i) numerous peptides bearing multiple lysine and/or arginine residues and (ii) identical portions of the protein sequence were found in multiple peptides. Most of these peptides were derived from five regions in transferrin. These results were interpreted to mean that proteolysis in the case of transferrin occurred faster than the rate at which buried lysine and arginine residues were unmasked in the five regions providing peptides that were only partially digested.     

Acid-labile surfactant improves in-sodium dodecyl sulfate polyacrylamide gel protein digestion for matrix-assisted laser desorption/ionization mass spectrometric peptide mapping

Mass spectrometry (MS) together with genome database searches serves as a powerful tool for the identification of proteins. In proteome analysis, mixtures of cellular proteins are usually separated by sodium dodecyl sulfate (SDS) polyacrylamide gel-based two-dimensional gel electrophoresis (2-DE) or one-dimensional gel electrophoresis (1-DE), and in-gel digested by a specific protease. In-gel protein digestion is one of the critical steps for sensitive protein identification by these procedures. Efficient protein digestion is required for obtaining peptide peaks necessary for protein identification by MS. This paper reports a remarkable improvement of protein digestion in SDS polyacrylamide gels using an acid-labile surfactant, sodium 3-[(2-methyl-2-undecyl-1,3-dioxolan-4-yl)methoxy]-1-propanesulfonate (ALS). Pretreatment of gel pieces containing protein spots separated by 2-DE with a small amount of ALS prior to trypsin digestion led to increases in the digested peptides eluted from the gels. Consistently, treatment of gel pieces containing silver-stained standard proteins and those separated from tissue extracts resulted in the detection of increased numbers of peptide peaks in spectra obtained by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOFMS). Hence the present protocol with ALS provides a useful strategy for sensitive protein identification by MS.     

Rapid fabrication of glass/PDMS hybrid µIMER for high throughput membrane proteomics

Mass spectrometry (MS) based proteomics has brought a radical approach to systems biology, offering a platform to study complex biological functions. However, key proteomic technical challenges remain, mainly the inability to characterise the complete proteome of a cell due to the thousands of diverse, complex proteins expressed at an extremely wide concentration range. Currently, high throughput and efficient techniques to unambiguously identify and quantify proteins on a proteome-wide scale are in demand. Miniaturised analytical systems placed upstream of MS help us to attain these goals. One time-consuming step in traditional techniques is the in-solution digestion of proteins (4-20 h). This also has other drawbacks, including enzyme autoproteolysis, low efficiency, and manual operation. Furthermore, the identification of α-helical membrane proteins has remained a challenge due to their high hydrophobicity and lack of trypsin cleavage targets in transmembrane helices. We demonstrate a new rapidly produced glass/PDMS micro Immobilised Enzyme Reactor (μIMER) with enzymes covalently immobilised onto polyacrylic acid plasma-modified surfaces for the purpose of rapidly (as low as 30 s) generating peptides suitable for MS analysis. This μIMER also allows, for the first time, rapid digestion of insoluble proteins. Membrane protein identification through this method was achieved after just 4 min digestion time, up to 9-fold faster than either dual-stage in-solution digestion approaches or other commonly used bacterial membrane proteomic workflows.     

Protein proteolysis and the multi-dimensional electrochromatographic separation of histidine-containing peptide fragments on a chip

This paper reports a system for three-dimensional electrochromatography in a chip format. The steps involved included trypsin digestion, copper(II)-immobilized metal affinity chromatography [Cu(II)-IMAC] selection of histidine-containing peptides, and reversed-phase capillary electrochromatography of the selected peptides. Trypsin digestion and affinity chromatography were achieved in particle-based columns with a microfabricated frit whereas reversed-phase separations were executed on a column of collocated monolithic support structures. Column frits were designed to maintain constant cross sectional area and path length in all channels and to retain particles down to a size of 3 microm. Cu(II)-IMAC selection of histidine-containing peptides from standard peptide mixtures and protein digests followed by reversed-phase chromatography of the selected peptides was demonstrated in the electrochromatography mode. The possibility to run a comprehensive proteomic analysis by combining trypsin digestion, affinity selection, and a reversed-phase separation on chips was shown using fluorescein isothiocyanate-labeled bovine serum albumin as an example.