Characterization of efficient proteolysis by trypsin loaded macroporous silica

Tryptic digestion of proteins in trypsin loaded porous silica has been shown to be highly efficient. Enzymatic silica-reactors were prepared by immobilizing trypsin into macroporous ordered siliceous foam (MOSF) and into mesoporous SBA-15 silica which has a smaller pore size. The tryptic products from the silica reactors were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), and a higher proteolysis efficiency was obtained with MOSF. These results can be well interpreted by a sequential digestion model taking into account the confinement and concentration enrichment of both the substrates and enzymes within the silica pores. Proteins at low concentrations and proteins in urea and surfactant solutions were also successfully digested with the MOSF-based reactor and identified by MS. Considering that the immobilized trypsin could retain its enzymatic activity for weeks, this MOSF reactor provides many advantages compared to free enzyme proteolysis. As a proof-of-concept, the digest of a real complex sample extracted from the cytoplasm of mouse liver tissue using trypsin loaded MOSF yielded better results than the typical in-solution protocol.     

Immobilization of trypsin on poly(urea-formaldehyde)-coated fiberglass cores in microchip for highly efficient proteolysis

Trypsin was covalently immobilized on poly(urea‐formaldehyde)‐coated fiberglass cores based on the condensation reaction between poly(urea‐formaldehyde) and trypsin for efficient microfluidic proteolysis in this work. Prior to use, a piece of the trypsin‐immobilized fiber was inserted into the main channel of a microchip under a magnifier to form a core‐changeable bioreactor. Because trypsin was not permanently immobilized on the channel wall, the novel bioreactor was regenerable. Two standard proteins, hemoglobin (HEM) and lysozyme (LYS), were digested by the unique bioreactor to demonstrate its feasibility and performance. The interaction time between the flowing proteins and the immobilized trypsin was evaluated to be less than 10 s. The peptides in the digests were identified by MALDI‐TOF MS to obtain PMF. The results indicated that digestion performance of the microfluidic bioreactor was better than that of 12‐h in‐solution digestion.     

On-chip microextraction for proteomic sample preparation of in-gel digests

Despite the high sensitivity and relatively high tolerance for contaminants of matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) there is often a need to purify and concentrate the sample solution, especially after in-gel digestion of proteins separated by two-dimensional gel electrophoresis (2-DE). A silicon microextraction chip (SMEC) for sample clean-up and trace enrichment of peptides was manufactured and investigated. The microchip structure was used to trap reversed-phase chromatography media (POROS R2 beads) that facilitates sample purification/enrichment of contaminated and dilute samples prior to the MALDI-TOF MS analysis. The validity of the SMEC sample preparation technique was successfully investigated by performing analysis on a 10 nM peptide mixture containing 2 m urea in 0.1 m phosphate-buffered saline with MALDI-TOF MS. It is demonstrated that the microchip sample clean-up and enrichment of peptides can facilitate identification of proteins from 2-DE separations. The microchip structure was also used to trap beads immobilized with trypsin, thereby effectively becoming a microreactor for enzymatic digestion of proteins. This microreactor was used to generate a peptide map from a 100 nM bovine serum albumin sample.     

Open Tubular Lab-On-Column/Mass Spectrometry for Targeted Proteomics of Nanogram Sample Amounts

A novel open tubular nanoproteomic platform featuring accelerated on-line protein digestion and high-resolution nano liquid chromatography mass spectrometry (LC-MS) has been developed. The platform features very narrow open tubular columns, and is hence particularly suited for limited sample amounts. For enzymatic digestion of proteins, samples are passed through a 20 µm inner diameter (ID) trypsin + endoproteinase Lys-C immobilized open tubular enzyme reactor (OTER). Resulting peptides are subsequently trapped on a monolithic pre-column and transferred on-line to a 10 µm ID porous layer open tubular (PLOT) liquid chromatography LC separation column. Wnt/ß-catenein signaling pathway (Wnt-pathway) proteins of potentially diagnostic value were digested+detected in targeted-MS/MS mode in small cell samples and tumor tissues within 120 minutes. For example, a potential biomarker Axin1 was identifiable in just 10 ng of sample (protein extract of ∼1,000 HCT15 colon cancer cells). In comprehensive mode, the current OTER-PLOT set-up could be used to identify approximately 1500 proteins in HCT15 cells using a relatively short digestion+detection cycle (240 minutes), outperforming previously reported on-line digestion/separation systems. The platform is fully automated utilizing common commercial instrumentation and parts, while the reactor and columns are simple to produce and have low carry-over. These initial results point to automated solutions for fast and very sensitive MS based proteomics, especially for samples of limited size.     

Preliminary 2-D chromatographic investigation of the human stem cell proteome

Stem cells represent a promising tool for the treatment of various hematopoietic diseases. In order to identify stem cell-specific proteins, the proteome of human stem cells from umbilical cord blood was explored for the first time. For this purpose, the crude lysate of 4 x 10(5) CD34+ cells was subjected to in solution trypsin digestion. The resulting peptides were then separated via cation exchange followed by reversed phase chromatography and analyzed by nanospray MS/MS. Database search revealed a total of 215 proteins which could be reliably identified. To obtain a more complete picture of the human stell cell proteome and to also access low abundant proteins, pooling of more than one CD34+ preparations seems necessary in order to increase the cell number and thus the protein content.     

Continuous free-flow electrophoresis separation of cytosolic proteins from the human colon carcinoma cell line LIM 1215: a non two-dimensional gel electrophoresis-based proteome analysis strategy

The conventional approach for analyzing the protein complement of a genome involves the combination of two-dimensional gel electrophoresis (2-DE) and mass spectrometric based protein identification technologies. While 2-DE is a powerful separation technique, it is severely limited by the insolubility of certain classes of proteins (e.g. hydrophobic membrane proteins), as well as the amount of protein that can be processed. Here, we describe a simple procedure for resolving complex mixtures of proteins that involves a combination of free flow electrophoresis (FFE), a liquid-based isoelectric focussing (IEF) method, and sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Resolved proteins were identified by peptide fragment sequencing using capillary column reversed-phase high performance liquid chromatography (RP-HPLC)/mass spectrometry (MS). An initial demonstration of the method was performed using digitonin/ethylenediaminetetraacetic acid EDTA extracted cytosolic proteins from the human colon carcinoma cell line, LIM 1215. Cytosolic proteins were separated by liquid-based IEF (pH range 3-10) into 96 fractions, and each FFE fraction was further fractionated by SDS-PAGE. Selected protein bands were excised from the SDS-PAGE gel, digested in situ with trypsin, and subsequently identified by on-line RP-HPLC/electrospray-ionization ion trap MS. Our results indicate that FFE is: (i) an extremely powerful liquid-based IEF method for resolving proteins; (ii) not limited by the amount of sample that can be loaded onto the instrument; and (iii) capable of fractionating intact protein complexes (a potentially powerful tool for cell-mapping proteomics). An up-to-date list of cytosolic proteins from the human colorectal carcinoma cell line LIM 1215 can be found in the Joint Protein Structure Laboratory (JPSL) proteome database. This information will provide an invaluable resource for future proteomics-based biological studies of colon cancer. The JPSL proteome database can be accessed through the World Wide Web (WWW) network (http://www.ludwig.edu.au/jpsl/jpslhome.html).     

Cell Surface Interactions between Trypanosoma congolense and Macrophages during Phagocytosis In Vitro

ABSTRACT. Trypanosoma congolense bloodstream forms preincubated with a high titer of anti-variant surface antigen (VSG)-specific antibody, a low amount of anti-VSG plus complement-active mouse serum (MS), MS alone, and trypsin were cocultivated with mouse peritoneal macrophages in vitro. Immunofluorescence as well as transmission and scanning electron microscopy revealed that upon attachment to the macrophages' surface, trypanosomes opsonized with anti-VSG/MS formed opsonized filopodia, which were rapidly internalized by the phagocytes. Although these cells attached as frequently as anti-VSG or trypsin-pretreated parasites, the rate of phagocytosis of anti-VSG/MS pretreated trypanosomes was reduced significantly. Trypanosomes pretreated with high antibody titers alone were lysed on the surface of the macrophages before phagocytosis was completed. Parasites opsonized with complement alone adhered only occasionally and were rarely phagocytosed. Trypsin-treated trypanosomes, which served as positive control cells, rapidly attached and remained intact until ingulfment by the macrophages was completed. Untreated control parasites did not attach to the macrophages and were not phagocytosed. Cocultivation of macrophages with anti-VSG/MS-opsonized trypanosomes caused internalization of the flagellum by membrane fusion. Filopodia formation by T. congolense is thus correlated with a marked reduction in phagocytosis even in the presence of only a sublytic antibody titer.     

Cell surface interactions between Trypanosoma congolense and macrophages during phagocytosis in vitro.

Trypanosoma congolense bloodstream forms preincubated with a high titer of anti-variant surface antigen (VSG)-specific antibody, a low amount of anti-VSG plus complement-active mouse serum (MS), MS alone, and trypsin were cocultivated with mouse peritoneal macrophages in vitro. Immunofluorescence as well as transmission and scanning electron microscopy revealed that upon attachment to the macrophages' surface, trypanosomes opsonized with anti-VSG/MS formed opsonized filopodia, which were rapidly internalized by the phagocytes. Although these cells attached as frequently as anti-VSG or trypsin-pretreated parasites, the rate of phagocytosis of anti-VSG/MS pretreated trypanosomes was reduced significantly. Trypanosomes pretreated with high antibody titers alone were lysed on the surface of the macrophages before phagocytosis was completed. Parasites opsonized with complement alone adhered only occasionally and were rarely phagocytosed. Trypsin-treated trypanosomes, which served as positive control cells, rapidly attached and remained intact until ingulfment by the macrophages was completed. Untreated control parasites did not attach to the macrophages and were not phagocytosed. Cocultivation of macrophages with anti-VSG/MS-opsonized trypanosomes caused internalization of the flagellum by membrane fusion. Filopodia formation by T. congolense is thus correlated with a marked reduction in phagocytosis even in the presence of only a sublytic antibody titer.     

Efficient on-chip proteolysis system based on functionalized magnetic silica microspheres

An easily replaceable enzymatic microreactor has been fabricated based on the glass microchip with trypsin-immobilized magnetic silica microspheres (MS microspheres). Magnetic microspheres with small size (approximately 300 nm in diameter) and high magnetic responsivity to magnetic field (68.2 emu/g) were synthesized and modified with tetraethyl orthosilicate (TEOS). Aminopropyltriethoxysilane (APTES) and glutaraldehyde (GA) were then introduced to functionalize the MS microspheres for enzyme immobilization. Trypsin was stably immobilized onto the MS microspheres through the reaction of primary amines of the proteins with aldehyde groups on the MS microspheres. The trypsin-immobilized MS microspheres were then locally packed into the microchannel by the application of a strong field magnet to form an on-chip enzymatic microreactor. The digestion efficiency and reproducibility of the microreactor were demonstrated by using cytochrome c (Cyt-C) as a model protein. When compared with an incubation time of 12 h by free trypsin in the conventional digestion approach, proteins can be digested by the on-chip microreactor in several minutes. This microreactor was also successfully applied to the analysis of an RPLC fraction of the rat liver extract. This opens a route for its further application in top-down proteomic analysis.     

Large-scale identification of Caenorhabditis elegans proteins by multidimensional liquid chromatography-tandem mass spectrometry

A proteome of a model organism, Caenorhabditis elegans, was analyzed by an integrated liquid chromatography (LC)-based protein identification system, which was constructed by microscale two-dimensional liquid chromatography (2DLC) coupled with electrospray ionization (ESI) tandem mass spectrometry (MS/MS) on a high-resolution hybrid mass spectrometer with an automated data analysis system. Soluble and insoluble protein fractions were prepared from a mixed growth phase culture of the worm C. elegans, digested with trypsin, and fractionated separately on the 2DLC system. The separated peptides were directly analyzed by on-line ESI-MS/MS in a data-dependent mode, and the resultant spectral data were automatically processed to search a genome sequence database, wormpep 66, for protein identification. The total number of proteins of the composite proteome identified in this method was 1,616, including 110 secreted/targeted proteins and 242 transmembrane proteins. The codon adaptation indices of the identified proteins suggested that the system could identify proteins of relatively low abundance, which are difficult to identify by conventional 2D-gel electrophoresis (GE) followed by an offline mass spectrometric analysis such as peptide mass fingerprinting. Among the approximately 5,400 peptides assigned in this study, many peptides with post-translational modifications, such as N-terminal acetylation and phosphorylation, were detected. This expression profile of C. elegans, containing 571 hypothetical gene products, will serve as the basic data of a major proteome set expressed in the worm.     

Comparative cytochrome P450 proteomics in the livers of immunodeficient mice using 18O stable isotope labeling

Quantitative changes in cytochrome P450 (CYP) proteins involved in drug metabolism as a consequence of drug treatment are important parameters in predicting the fates and pharmacological consequences of xenobiotics and drugs. In this study we undertook comparative P450 proteomics using liver from control and 1,4-bis-2-(3,5-dichloropyridyloxybenzene) (TCPOBOP)-dosed mice. The method involved separation of microsomal proteins by SDS-PAGE, trypsin digestion, and postdigest 18O/16O labeling followed by nano-LC-MS/MS for peptide identification and LC-MS for relative quantification. Seventeen P450 proteins were identified from mouse liver of which 16 yielded data sufficient for relative quantification. All the P450s detected were unambiguously identified except the highly homologous CYP2A4/2A5. With the exception of CYP2A12, -2D10, and -2F2, the levels of all the P450s quantified were affected by treatment with TCPOBOP (3 mg/kg). CYP1A2, -2A4/5, -2B10, -2B20, -2C29, -2C37, -2C38, -3A11, and -39A1 were up-regulated, and CYP2C40, -2E1, -3A41, and -27A1 down-regulated. The response of CYP2B20 to stimulation has not been distinguished previously from that of CYP2B10 because of the poor discrimination between these two proteins (they share 87% sequence identity). Differential response to chemical stimulation by closely related members of the CYP2C subfamily was also observed.     

Hydrophilic polydopamine‐coated magnetic graphene nanocomposites for highly efficient tryptic immobilization

In this work, polydopamine‐coated magnetic graphene (MG@PDA) nanocomposites were synthesized by a facile method. Trypsin was then directly immobilized on the surface of the nanocomposites through simple PDA chemistry with no need for introducing any other coupling groups. The as‐made MG@PDA nanocomposites inherit not only the large surface area of graphene which makes them capable of immobilizing high amount of trypsin (up to 0.175 mg/mg), but also the good hydrophilicity of PDA which greatly improves their biocompatibility. Moreover, the strong magnetic responsibility makes them easy to be separated from the digested peptide solution when applying a magnetic field. The feasibility of the trypsin‐immobilized MG@PDA (MG@PDA‐trypsin) nanocomposites for protein digestion was investigated and the results indicated their high digestion efficiency in a short digestion time (10 min). In addition, the reusability and stability of the MG@PDA‐trypsin nanocomposites were also tested in our work. To further confirm the efficiency of MG@PDA‐trypsin nanocomposites for proteome analysis, they were applied to digest proteins extracted from skimmed milk, followed by nano RPLC‐ESI‐MS/MS analysis, and a total of 321 proteins were identified, much more than those obtained by 16‐h in‐solution digestion (264 proteins), indicating the great potential of MG@PDA‐trypsin nanocomposites as the supports for high‐throughput proteome study.     

Quartz crystal biosensor for real-time monitoring of molecular recognition between protein and small molecular medicinal agents

A quartz crystal microbalance (QCM) biosensor integrated into a flow injection analysis (FIA) system was used for the real-time investigation of molecular recognition between a protein and small molecular medicinal agents. Two sulfa-drugs, sulfamethazine (SMZ) and sulfamethoxazole (SMO), were, respectively, immobilized on the gold electrodes of the piezoelectric crystals using appropriate procedures based on self-assembly of the dithiothreitol (DTT). The binding interactions of the two immobilized drug ligands, with various proteins in solution, were followed as changes in the resonant frequency of the modified crystals. Results obtained from this rapid screen analysis clearly indicated that the two drug ligands appeared quite different in this molecular recognition procedure although their structures were similar. SMZ-immobilized sensor showed specific interaction only with IgG, while SMO-immobilized sensor showed negligible specific binding with IgG, but binding with trypsin and chymotrypsin. Further studies on the specific interaction between immobilized SMZ and three different species of IgG--human IgG, goat IgG and mouse IgG were carried out and the marked species-dependent difference was observed. The resultant sensorgrams were rapidly analyzed by using an in-house kinetic analysis software based on genetic algorithm (GA) to derive both the kinetic rate constants (kass and kdiss) and equilibrium association constants (KA) for IgG-SMZ interactions. For the interactions, KA were 5.48 x 10(5), 2.75 x 10(5) and 1.86 x 10(5) M(-1) for human IgG, goat IgG and mouse IgG, respectively. The kinetic data provided further insight into the structural/functional relationships of different IgG on a molecular level.     

A systematical analysis of tryptic peptide identification with reverse phase liquid chromatography and electrospray ion trap mass spectrometry

In this study we systematically analyzed the elution condition of tryptic peptides and the characteristics of identified peptides in reverse phase liquid chromatography and electrospray tandem mass spectrometry (RPLC-MS/MS) analysis. Following protein digestion with trypsin, the peptide mixture was analyzed by on-line RPLC-MS/MS. Bovine serum albumin (BSA) was used to optimize acetonitrile (ACN) elution gradient for tryptic peptides, and Cytochrome C was used to retest the gradient and the sensitivity of LC-MS/MS. The characteristics of identified peptides were also analyzed. In our experiments, the suitable ACN gradient is 5% to 30% for tryptic peptide elution and the sensitivity of LC-MS/MS is 50 fmol.Analysis of the tryptic peptides demonstrated that longer (more than 10 amino acids) and multi-charge state ( 2, 3) peptides are likely to be identified, and the hydropathicity of the peptides might not be related to whether it is more likely to be identified or not. The number of identified peptides for a protein might be used to estimate its loading amount under the same sample background. Moreover, in this study the identified peptides present three types of redundancy, namely identification, charge, and sequence redundancy, which may repress low abundance protein identification.     

A strategy for high-resolution protein identification in surface-enhanced laser desorption/ionization mass spectrometry: calgranulin A and chaperonin 10 as protein markers for endometrial carcinoma

Surface-enhanced laser desorption/ionization-mass spectrometry (SELDI-MS) has conventionally been practiced on linear time of flight (TOF) which has low mass accuracy and resolution. Here we demonstrate in an examination of both malignant and nonmalignant endometrial tissue homogenates that high mass accuracy and resolution in the MS stage are crucial. Using a commercially available quadrupole/TOF (QqTOF), we were able to resolve two potential cancer markers, subsequently identified off-line as chaperonin 10 and calgranulin A, that differ by 8 Da in mass. Two off-line protein identification protocols were developed: the first was based on size-exclusion chromatography (SEC), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), protein extraction, trypsin digestion, and matrix-assisted laser desorption/ionization-tandem MS (MALDI-MS/MS); the second on SEC and shotgun nano-liquid chromatography (nanoLC)-MS/MS. Analyses on a cohort of 44 endometrial homogenates showed 22 out of 23 nonmalignant samples had nondetectable to very low abundance of chaperonin 10 and calgranulin A; 17 of the 21 malignant samples had detectable to abundant levels of both proteins. Immunohistochemical staining of a tissue microarray of 32 samples showed that approximately half of malignant endometrial tissues exhibited positive staining for calgranulin A in the malignant epithelium, while 9 out of 10 benign tissues exhibited negative epithelial staining. In addition, macrophages/granulocytes in malignant as well as nonmalignant tissues showed positive staining. No immunostaining occurred in stroma or myometrium. Calgranulin A, in combination with chaperonin 10 and other proteins, may eventually constitute a panel of markers to permit diagnosis and screening of endometrial cancer.     

Lumbar cerebrospinal fluid proteome in multiple sclerosis: characterization by ultrafiltration, liquid chromatography, and mass spectrometry

Neurological diseases, including multiple sclerosis (M.S.), often provoke changes in the functioning of the endothelial and epithelial brain barriers and give rise to disease-associated alterations of the cerebrospinal fluid (CSF) proteome. In the present study, pooled and ultrafiltered CSF of M.S. and non-M.S. patients was digested with trypsin and analyzed by off-line strong cation-exchange chromatography (SCX) coupled to on-line reversed-phase LC-ESI-MS/MS. In an alternative approach, the trypsin-treated subproteomes were analyzed directly by LC-ESI-MS/MS and gas-phase fractionation in the mass spectrometer. Taken together, both proteomic approaches in combination with a three-step evaluation process including the search engines Sequest and Mascot, and the validation software Scaffold, resulted in the identification of 148 proteins. Sixty proteins were identified in CSF for the first time by mass spectrometry. For validation purposes, the concentration of cystatin A was determined in individual CSF and serum samples of M.S. and non-M.S. patients using ELISA.     

A novel trypsin inhibitor from Peltophorum dubium seeds,with lectin-like properties,triggers rat lymphoma cell apoptosis

A trypsin inhibitor (PDTI) was isolated from Peltophorum dubium seeds by affinity chromatography on a thyroglobulin-agarose or a trypsin-agarose column. In both cases, SDS-PAGE showed two bands of M(r) 20,000 and 22,000, which could not be resolved. Their amino-terminal sequences were identical and similar to that of Kunitz-type soybean trypsin inhibitor (SBTI). Mass spectrometry analysis of tryptic digests of both bands showed 16 coincident peaks, suggesting that they are closely related proteins. The K(i)s for trypsin and chymotrypsin inhibitory activity of PDTI were 1.6 x 10(-7) and 1.3 x 10(-5)M, respectively. Lectin-like activity of PDTI and SBTI, detected by hemagglutination of rabbit erythrocytes, was inhibited by sialic acid-containing compounds. PDTI and SBTI caused apoptosis of Nb2 rat lymphoma cells, demonstrated by decrease of viability, DNA hypodiploidy, DNA fragmentation, and caspase-3-like activity. They had no effect on normal mouse splenocytes or lymphocytes, whereas they caused apoptosis of concanavalin A-stimulated mouse lymphocytes.     

Proteomic analysis of mouse brain microsomes: identification and bioinformatic characterization of endoplasmic reticulum proteins in the mammalian central nervous system

The endoplasmic reticulum (ER) is the main source for the storage and release of intracellular calcium in neurons and, thus, contributes to the functionality of a diverse set of pathways that control critical aspects of central nervous system function including but not limited to gene expression, neurotransmission, learning, and memory. ER-derived proteins obtained after subcellular fractionation of mouse brain homogenate were digested with trypsin and the corresponding peptides fractionated by strong cation exchange chromatography followed by LC-MS/MS analysis on a hybrid linear ion trap--Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. A comprehensive catalogue representing 1914 proteins was generated from this particular proteomic analysis using identification criteria that corresponded to a false positive identification rate of 0.4%. Various molecular functions and biological processes relevant to the ER were identified upon gene ontology (GO)-based analysis including pathways associated with molecular transport, protein trafficking and localization, and cell signaling. Comparison of the 2D-LC-MS/MS results with those obtained from shotgun LC-MS/MS analyses demonstrated that most molecular functions and biological processes were represented via GO analysis using either methodology. Results from this comparison as well as a focused investigation into components of calcium-mediated signaling in the mouse brain ER are also presented.