Microcolumn capture and digestion of proteins combined with mass spectrometry for protein identification

A procedure has been developed for protein identification using mass spectrometry (MS) that incorporates sample cleanup, preconcentration, and protein digestion in a single-stage system. The procedure involves the adsorption of a protein, or protein mixture, from solution onto a hydrophobic resin that is contained within a microcolumn. Sample loading is accomplished by flowing the protein solution through the microcolumn, where the protein adsorbs to the hydrophobic surface. The protein is digested while still bound to the hydrophobic surface by flowing a buffered trypsin solution through the column bed. The peptide fragments are subsequently eluted for detection by MALDI or ESI-MS. The procedure is demonstrated using dilute protein samples containing high concentrations of salt, urea, and modest amount of sodium dodecyl sulfate relative to protein. Peptide fragments are also detected by MS from a 500 nM bacteriorhodopsin solution digested in a microcolumn. In this case, a combined cyanogen bromide/trypsin digestion was performed in-column. The procedure is applied to the MALDI-MS/MS identification of proteins present in an individual fraction collected by ion exchange HPLC separation of E. coli total cell extract. An additional application is illustrated in the analysis of a human plasma fraction. A total of 14 proteins, which were present in the sample at sub-micromolar concentrations, were identified from ESI-MS/MS. The microcolumn digestion procedure represents the next step toward a system for fully automated protein analysis through capture and digestion of the adsorbed protein on hydrophobic surfaces.     

Isolation and characterization of two major serum proteins from the dogfish, Mustelus canis, C-reactive protein and amyloid P component

Two major serum components from the dogfish, Mustelus canis, have been isolated using affinity chromatography. Both proteins bind to an AH-Sepharose 4B-phosphorylcholine affinity matrix in the presence of Ca2+ and are eluted from the column by EDTA. Upon readdition of Ca2+ to the eluted proteins, the two proteins can be separated by passage through a column of Sepharose CL-4B. The first protein, C-reactive protein, passes through the Sepharose CL-4B column in the presence of Ca2+ whereas the second protein, serum amyloid P component, remains bound. The serum amyloid P component is then eluted from the Sepharose CL-4B in pure form by EDTA. The dogfish C-reactive protein isolated by the phosphorylcholine affinity matrix precipitates with pneumococcal C-polysaccharide and with a synthetic derivative of bovine serum albumin to which phosphorylcholine is covalently attached. The precipitation is inhibited by either EDTA or by phosphorylcholine. Dogfish C-reactive protein has a molecular weight of approximately 250,000 with dimeric subunits of Mr = 50,000. Upon addition of beta-mercaptoethanol these dimeric subunits dissociate to two identical monomeric subunits of Mr = 25,000. The protein cross-reacts immunologically with goat antisera prepared against rabbit C-reactive protein. The dogfish serum amyloid P component has a molecular weight of at least 250,000 with monomeric subunits of Mr = 25,000. Cross-reactivity of the amyloid P component with the C-reactive protein could not be shown. However, NH2-terminal sequence analysis of the first 20 amino acids showed some homology. The relationship of dogfish C-reactive protein to the C-reactive proteins in Limulus polyphemus and in rabbits and humans is discussed.     

Binding proteins for sweet compounds from gustatory papillae of the cow, pig and rat

The intensely sweet proteins thaumatin and monellin were covalently attached to affinity column supports. Lingual tissue extracts were incubated with the affinity columns which were then eluted with glycine-HCl pH 3.4, the sweet peptide aspartame, or gymnemic acid, which is a sweet taste modifier. SDS-PAGE analysis of eluates from the columns showed that 156 kDa and 47 kDa proteins were the main components from cow fungiform papillae which were specifically bound to thaumatin and monellin. These proteins could be displaced from the column with 0.5 mM aspartame or 0.5 mg/ml gymnemic acid. With circumvallate papillae small amounts of 47 kDa protein were also found. The 47 kDa protein was also the major component bound to a gymnemic acid affinity column and could be displaced from the column with 0.5 mg/ml gymnemic acid. Control experiments with other lingual tissue components indicated that these proteins are localised in the gustatory papillae. Similar protein patterns were also found in extracts of pig fungiform papillae and rat lingual preparations.     

Induced binding of proteins by ammonium sulfate in affinity and ion-exchange column chromatography

In general, proteins bind to affinity or ion-exchange columns at low salt concentrations, and the bound proteins are eluted by raising the salt concentration, changing the solvent pH, or adding competing ligands. Blue-Sepharose is often used to remove bovine serum albumin (BSA) from samples, but when we applied BSA to Blue-Sepharose in 20 mM phosphate, pH 7.0, 50%-60% of the protein flowed through the column; however, complete binding of BSA was achieved by the addition of 2 M ammonium sulfate (AS) to the column equilibration buffer and the sample. The bound protein was eluted by decreasing the AS concentration or by adding 1 M NaCl or arginine. AS at high concentrations resulted in binding of BSA even to an ion-exchange column, Q-Sepharose, at pH 7.0. Thus, although moderate salt concentrations elute proteins from Blue-Sepharose or ion-exchange columns, proteins can be bound to these columns under extreme salting-out conditions. Similar enhanced binding of proteins by AS was observed with an ATP-affinity column.     

The proteomic reactor: a microfluidic device for processing minute amounts of protein prior to mass spectrometry analysis

Gel-free proteomics has emerged as a complement to conventional gel-based proteomics. Gel-free approaches focus on peptide or protein fractionation, but they do not address the efficiency of protein processing. We report the development of a microfluidic proteomic reactor that greatly simplifies the processing of complex proteomic samples by combining multiple proteomic steps. Rapid extraction and enrichment of proteins from complex proteomic samples or directly from cells are readily performed on the reactor. Furthermore, chemical and enzymatic treatments of proteins are performed in 50 nL effective volume, which results in an increased number of generated peptides. The products are compatible with mass spectrometry. We demonstrated that the proteomic reactor is at least 10 times more sensitive than current gel-free methodologies with one protein identified per 440 pg of protein lysate injected on the reactor. Furthermore, as little as 300 cells can be directly introduced on the proteomic reactor and analyzed by mass spectrometry.     

Mass-spectrometry-linked screening of protein fractions for enzymatic activities--a tool for functional genomics

A simple and rapid strategy is described to screen protein fractions for defined enzymatic activity. A protein fraction from a porcine kidney extract was immobilized by covalent coupling to activated affinity beads. The immobilized proteins were incubated with probes specific for different enzyme activities. The reaction products were analyzed by matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry. The MALDI spectra indicate the presence of 5'-nucleotidase, phosphatase, kinase, glutathione reductase, and renin activities in the kidney protein extract. Furthermore, the method can be used to screen for inhibitors of enzymatic reactions. The method is adaptable to high-throughput sample handling and automated mass spectrometric analysis and therefore suited for functional genomics.     

Mass-Spectrometry-Linked Screening of Protein Fractions for Enzymatic Activities—A Tool for Functional Genomics

A simple and rapid strategy is described to screen protein fractions for defined enzymatic activity. A protein fraction from a porcine kidney extract was immobilized by covalent coupling to activated affinity beads. The immobilized proteins were incubated with probes specific for different enzyme activities. The reaction products were analyzed by matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry. The MALDI spectra indicate the presence of 5′-nucleotidase, phosphatase, kinase, glutathione reductase, and renin activities in the kidney protein extract. Furthermore, the method can be used to screen for inhibitors of enzymatic reactions. The method is adaptable to high-throughput sample handling and automated mass spectrometric analysis and therefore suited for functional genomics.     

The proteomic reactor facilitates the analysis of affinity-purified proteins by mass spectrometry: application for identifying ubiquitinated proteins in human cells

Mass spectrometry (MS) coupled to affinity purification is a powerful approach for identifying protein-protein interactions and for mapping post-translational modifications. Prior to MS analysis, affinity-purified proteins are typically separated by gel electrophoresis, visualized with a protein stain, excised, and subjected to in-gel digestion. An inherent limitation of this series of steps is the loss of protein sample that occurs during gel processing. Although methods employing in-solution digestion have been reported, they generally suffer from poor reaction kinetics. In the present study, we demonstrate an application of a microfluidic processing device, termed the Proteomic Reactor, for enzymatic digestion of affinity-purified proteins for liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. Use of the Proteomic Reactor enabled the identification of numerous ubiquitinated proteins in a human cell line expressing reduced amounts of the ubiquitin-dependent chaperone, valosin-containing protein (VCP). The Proteomic Reactor is a novel technology that facilitates the analysis of affinity-purified proteins and has the potential to aid future biological studies.     

Induced binding of proteins by ammonium sulfate in affinity and ion-exchange column chromatography

In general, proteins bind to affinity or ion-exchange columns at low salt concentrations, and the bound proteins are eluted by raising the salt concentration, changing the solvent pH, or adding competing ligands. Blue-Sepharose is often used to remove bovine serum albumin (BSA) from samples, but when we applied BSA to Blue-Sepharose in 20 mM phosphate, pH 7.0, 50%–60% of the protein flowed through the column; however, complete binding of BSA was achieved by the addition of 2 M ammonium sulfate (AS) to the column equilibration buffer and the sample. The bound protein was eluted by decreasing the AS concentration or by adding 1 M NaCl or arginine. AS at high concentrations resulted in binding of BSA even to an ion-exchange column, Q-Sepharose, at pH 7.0. Thus, although moderate salt concentrations elute proteins from Blue-Sepharose or ion-exchange columns, proteins can be bound to these columns under extreme salting-out conditions. Similar enhanced binding of proteins by AS was observed with an ATP-affinity column.     

Purification from goat antiserum of immunoglobulins against G6PD from rabbits]

DEAE Affi-Gel Blue (Bio-Rad) provides an efficient and rapid fractionation of human serum proteins by a single chromatographic step. When goat serum is applied to the matrix and chromatography is performed following the procedure utilized for the human serum proteins, the elution pattern changes and the Ig purification is not satisfactory. We achieved a better Ig purification from goat serum by the following improved procedure. We performed first an AS-40 fractionation followed by extensive dialysis in 50 mM Na-citrate pH 5.7. The sample was then loaded onto a P11 column equilibrated in the same buffer. The fraction eluted at Vo contained total IgG and the other serum proteins, except beta-globulins which were eluted with 0.24 M phosphate. Peak 1 concentrated and dialyzed in 20 mM phosphate buffer pH 8 was then applied to a DEAE Affi-Gel Blue column, equilibrated in the same buffer. Two protein peaks were eluted from this column and electrophoretically characterized as: peak 1, containing a pure Ig fraction (70% yield), peak 2 with albumin and other contaminating serum proteins. When goat antiserum is obtained against a specific protein, our technique may be suitably employed to purify polyclonal antibodies for immunoprecipitation studies.     

Centrifugal precipitation chromatography: principle, apparatus, and optimization of key parameters for protein fractionation by ammonium sulfate precipitation

A novel chromatographic system introduced here internally generates a concentration gradient of ammonium sulfate (AS) through a long separation channel under a centrifugal force field. Protein samples are exposed to a gradually increasing AS concentration and precipitated along the channel. Then, chromatographic elution is initiated by gradually decreasing the AS concentration in the gradient which causes the proteins to repeat dissolution and precipitation through the channel. Consequently, they are eluted out in the order of their solubility in the AS solution. The separation column consists of a pair of disks equipped with mutually mirror-imaged spiral grooves. A dialysis membrane is sandwiched between the disks to form two identical channels partitioned by the membrane. The disk assembly is mounted on the sealless continuous-flow centrifuge. When a concentrated AS solution is eluted through one channel and water through the other channel in an opposite direction, an exponential AS gradient is formed through the water channel. A series of basic experiments was performed to study the rates of AS transfer and osmosis through the membrane, and the operational parameters including elution time, revolution speed, inclination of gradient, and sample size were optimized using stable protein samples. Preliminary applications were successful in purification of monoclonal antibody from cell culture supernatant and an affinity separation of recombinant ketosteroid isomerase from a crude Escherichia coli lysate.     

Affinity chromatography of protein kinase C-phorbol ester receptor on polyacrylamide-immobilized phosphatidylserine

An affinity column, prepared by immobilizing phosphatidylserine and cholesterol in polyacrylamide, was utilized in the purification of protein kinase C. Protein kinase activity and phorbol ester binding were monitored by assaying Ca2+ plus phosphatidylserine-dependent phosphorylation of histone H1 and [3H]phorbol dibutyrate binding, respectively. Both activities were present in a cytosolic extract of rabbit renal cortex, eluted together from a DEAE-cellulose column, bound to the affinity column in the presence of Ca2+, and eluted symmetrically upon application of EGTA. Recovery from the affinity column was high (30-50%) and resulted in as much as a 6000-7700-fold purification, depending on the region of the DEAE-cellulose peak that was applied. Following affinity column purification, protein kinase and phorbol ester binding activity eluted symmetrically upon gel filtration, with a molecular weight of approximately 80 kDa. A protein of the same size was present in silver-stained gels following sodium dodecyl sulfate-polyacrylamide gel electrophoresis of affinity column purified samples from the DEAE-cellulose peak. From 2-4 other, smaller proteins were also present, their number and relative amounts depending on the region of the DEAE-cellulose peak used. These data indicate that Ca2+-dependent/binding to a polyacrylamide-immobilized phospholipid provides a useful technique for purification of protein kinase C as well as other, unidentified proteins exhibiting a Ca2+ plus phospholipid-dependent interaction.     

Quantification by affinity perfusion chromatography of phosphorylated BRCAl and BRCA2 proteins from tumor cells after lycopene treatment

A new procedure for the quantification of phosphorylated BRCA1 (P-BRCA1) and BRCA2 (P-BRCA2) proteins in breast cell lines after different treatments was carried out. Cells were cultivated with [35S]-methionine and extracts subjected to three perfusion chromatographies. First heparin affinity chromatography purified cellular DNA-binding proteins. Subsequent specific immunoprecipitation of BRCA1 and BRCA2 proteins was performed with antibodies raised against BRCA1 or BRCA2. The immune complexes were isolated by protein A affinity chromatography. Phosphorylated BRCA1 or BRCA2 proteins were then purified with a Poros 20 AL column where anti-phosphothreonine and anti-phosphoserine antibodies were previously bound. The percentage of phosphorylated BRCA1 or BRCA2 proteins was calculated as follows: 100 x dpm of P-BRCA1 or P-BRCA2 eluted from the POROS 20AL column/total dpm eluted from POROS 20AL column. Treatment with 10 microM lycopene increased P-BRCA1 and P-BRCA2 in the breast tumor cell line MCF7 but not in MDA-MB-231 or MCF-10a, breast tumor or fibrocystic cell lines, respectively.     

Assessment of albumin removal from an immunoaffinity spin column: Critical implications for proteomic examination of the albuminome and albumin‐depleted samples

High abundance proteins in serum and plasma (e.g., albumin) are routinely removed during proteomic sample processing as they can mask lower abundance proteins and peptides of biological/clinical interest. A common method of albumin depletion is based on immunoaffinity capture, and many immunoaffinity devices are designed for multiple uses. In this case, it is critical that the albumin captured on the affinity matrix is stripped from the column prior to regeneration of the matrix and processing of subsequent samples, to ensure no carryover and that maximal binding sites are available for subsequent samples. The current study examines the ability of a manufacturer's protocol to remove the proteins and peptides captured by an immunoaffinity spin column. The data presented in the current work illustrate the difficulty in completely removing albumin from the immunoaffinity device, and consequently, may explain the variability and decreased efficiency shown for this device in previous studies. In summary, the current data present important considerations for the implementation of multiple‐use immunoaffinity devices for processing subsequent clinical samples in a proteomic workflow.     

In situ fabrication of a microfluidic device for immobilised metal affinity sensing

In this work a novel microfluidic device was constructed in situ containing the smallest microscopic co-polymeric immobilised metal affinity (IMA) adsorbent yet documented. This device has for the first time allowed the microlitre scale chromatographic assay of histidine-tagged proteins in a biological sample. To enable this approach, rather than using a high capacity commercial packed bed column which requires large sample volumes and would be susceptible to occlusion by cell debris, a microgram capacity co-polymeric chromatographic substrate suitable for analytical applications was fabricated within a microfluidic channel. This porous co-polymeric IMA micro-chromatographic element, only 27μl in volume, was assessed for the analytical capture of two different histidine-tagged recombinant fusion proteins. The micro-chromatographic adsorber was fabricated in situ by photo-polymerising an iminodiacetic acid (IDA) functionalised polymer matrix around a template of fused 100μm diameter NH(4)Cl particles entirely within the microfluidic channel and then etching away the salt with water to form a network of interconnected voids. The surface of the micro-chromatographic adsorber was chemically functionalised with a chelating agent and loaded with Cu(2+) ions. FTIR and NMR analysis verified the presence of the chelating agent on the adsorbent surface and its Cu(2+) ion binding capacity was determined to be 2.4μmol Cu(2+) (ml of adsorbent)(-1). Micro-scale equilibrium adsorption studies using the two different histidine-tagged proteins, LacI-His(6)-GFP and α-Synuclein-His(8)-YFP, were carried out and the protein binding capacity of the adsorbent was determined to be 0.370 and 0.802mg(g of adsorbent)(-1), respectively. The dynamic binding capacity was determined at four different flow rates and found to be comparable to the equilibrium binding capacity at low flow rates. The sensing platform was also used to adsorb LacI-His(6)-GFP protein from crude cell lysate. During adsorption, laser scanning confocal microscopy identified locations within the adsorbent where protein adsorption and desorption occurred. The findings indicate that minimal channelling, selective product capture and near quantitative elution of the captured (adsorbed) product could be achieved, supporting the application of this new device as a high-throughput process analytical tool (PAT) for the in-process monitoring of histidine-tagged proteins in manufacturing.     

Mapping protein cysteine sulfonic acid modifications with specific enrichment and mass spectrometry: An integrated approach to explore the cysteine oxidation

Oxidation of thiol proteins, which results in conversion of cysteine residues to cysteine sulfenic, sulfinic or sulfonic acids, is an important posttranslational control of protein function in cells. To facilitate the analysis of this process with MALDI‐MS, we have developed a method for selective enrichment and identification of peptides containing cysteine sulfonic acid (sulfopeptides) in tryptic digests of proteins based on ionic affinity capture using polyarginine‐coated nanodiamonds as high‐affinity probes. The method was applied to selectively concentrate sulfopeptides from either a highly dilute solution or a complex peptide mixture in which the abundance of the sulfonated analyte is as low as 0.02%. The polyarginine‐coated probes exhibit a higher affinity for peptides containing multiple sulfonic acids than peptides containing single sulfonic acid. The limit of the detection is in the femtomole range, with the MALDI‐TOF mass spectrometer operating in the negative ion mode. The results show that the new approach has good specificity even in the presence of phosphopeptides. An application of this method for selective enrichment and structural identification of sulfopeptides is demonstrated with the tryptic digests of performic‐acid‐oxidized BSA.     

A microfluidic device for multiplexed protein detection in nano-liter volumes

We describe a microfluidic immunoassay device that permits sensitive and quantitative multiplexed protein measurements on nano-liter-scale samples. The device exploits the combined power of integrated microfluidics and optically encoded microspheres to create an array of approximately 100-μm² sensors functionalized with capture antibodies directed against distinct targets. This strategy overcomes the need for performing biochemical coupling of affinity reagents to the device substrate, permits multiple proteins to be detected in a nano-liter-scale sample, is scalable to large numbers of samples, and has the required sensitivity to measure the abundance of proteins derived from single mammalian cells. The sensitivity of the device is sufficient to detect 1000 copies of tumor necrosis factor (TNF) in a volume of 4.7 nl.     

Specific on-plate enrichment of phosphorylated peptides for direct MALDI-TOF MS analysis

An on-plate specific enrichment method is presented for the direct analysis of peptides phosphorylation. An array of sintered TiO 2 nanoparticle spots was prepared on a stainless steel plate to provide porous substrate with a very large specific surface and durable functions. These spots were used to selectively capture phosphorylated peptides from peptide mixtures, and the immobilized phosphopeptides could then be analyzed directly by MALDI MS after washing away the nonphosphorylated peptides. beta-Casein and protein mixtures were employed as model samples to investigate the selection efficiency. In this strategy, the steps of phosphopeptide capture, purification, and subsequent mass spectrometry analysis are all successfully accomplished on a single target plate, which greatly reduces sample loss and simplifies analytical procedures. The low detection limit, small sample size, and rapid selective entrapment show that this on-plate strategy is promising for online enrichment of phosphopeptides, which is essential for the analysis of minute amount of samples in high-throughput proteome research.     

Targeted protein pullout from human tissue samples using competitive elution

One commonly used strategy to gain information on the proteins in a cell is to isolate the proteins of interest by specific binders, often antibodies. Not only the specificity of the capturing antibodies but also the washing and elution conditions are crucial to avoid false-positive protein identifications. Eluting the target protein from the matrix, while avoiding the release of unrelated background proteins, should both provide more correct information on the target protein and its interaction partners, and minimize the effort to perform downstream analyses through the reduced number of eluted proteins. In this study, a novel approach for selective protein pullout is presented. Monospecific antibodies were used to selectively pullout target proteins from a complex biosample. Subsequently, the target proteins were competitively eluted from the affinity media with the recombinant antigen. To deplete the antigen from the eluted sample, IMAC spin columns were utilized to bind the N-terminal His-tag of the antigens. The competitive elution method was applied both to a model system, and for the extraction of a native human target protein. In the model system the recombinant target protein BBC7 was spiked into a protein extract of human liver, whereas an endogenously expressed target protein, cTAGE5, was extracted from the liver extract directly. SDS-PAGE analysis and mass spectrometry confirmed affinity isolation of expected target proteins. More selective elution was obtained using the competitive procedure as compared to elution at low pH. Competitive elution has thus been shown to offer an effective approach for wide-scale pullout experiments where proteins and their interaction partners are to be studied.     

Preparation and characterization of nitrilotriacetic-acid-terminated self-assembled monolayers on gold surfaces for matrix-assisted laser desorption ionization-time of flight-mass spectrometry analysis of proteins and peptides

On-target affinity capture, enrichment and purification of biomolecules improve detection of specific analytes from complex biological samples in matrix-assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS) analysis. In this paper, we report a simple method for preparation of a self-assembled nitrilotriacetic acid (NTA) monolayer on gold surface which can be used as a MALDI-TOF-MS sample target specifically for recombinant oligohistidine-tagged proteins/peptides and phosphorylated peptides. The NTA functional groups are immobilized to the gold surface via the linkage of 1,8-octanedithiol which forms a self-assembled monolayer on gold. Characterization by X-ray photoelectron spectroscopy and MALDI analysis of the modified surface are described. The chemically modified surface shows strong affinity toward the analytes of interest, which allows effective removal of the common interferences, e.g. salts and detergents, and therefore leads to improved signal/noise ratio and detection limit. The use of the modified surface simplifies the sample preparation for MALDI analysis of these targeted analytes.