Probing adenosine nucleotide-binding proteins with an affinity-labeled nucleotide probe and mass spectrometry

Mass spectrometry combined with chemical labeling strategies has become very important in biological analysis. Herein, we described the application of a biotin-conjugated acyl nucleotide for probing adenosine nucleotide-binding proteins. We demonstrated that the probe reacted specifically with the lysine residue at the nucleotide-binding site of two purified adenosine nucleotide-binding proteins, Escherichia coli recombinase A (RecA) and Saccharomyces cerevisiae alcohol dehydrogenase-I (YADH-I). A single conjugate peptide with a specifically labeled lysine residue was identified, by using LC-MS/MS, from the tryptic digestion mixture of the reaction products of the nucleotide analogue with RecA or YADH-I. The strategy, which involved labeling reaction, enzymatic digestion, affinity purification, and LC-MS/MS analysis, was relatively simple, fast, and straightforward. The method should be generally applicable for the identification of lysine residues at the nucleotide-binding site of other proteins. The biotin-conjugated acyl nucleotide probe also allowed for the enrichment and identification of nucleotide-binding proteins from complex protein mixtures; we showed that more than 50 adenosine nucleotide-binding proteins could be identified from the whole-cell lysates of HeLa-S3 and WM-266-4 cells.     

Integration of isoelectric focusing with parallel sodium dodecyl sulfate gel electrophoresis for multidimensional protein separations in a plastic microfluidic [correction of microfludic] network

An integrated protein concentration/separation system, combining non-native isoelectric focusing (IEF) with sodium dodecyl sulfate (SDS) gel electrophoresis on a polymer microfluidic chip, is reported. The system provides significant analyte concentration and extremely high resolving power for separated protein mixtures. The ability to introduce and isolate multiple separation media in a plastic microfluidic network is one of two key requirements for achieving multidimensional protein separations. The second requirement lies in the quantitative transfer of focused proteins from the first to second separation dimensions without significant loss in the resolution acquired from the first dimension. Rather than sequentially sampling protein analytes eluted from IEF, focused proteins are electrokinetically transferred into an array of orthogonal microchannels and further resolved by SDS gel electrophoresis in a parallel and high-throughput format. Resolved protein analytes are monitored using noncovalent, environment-sensitive, fluorescent probes such as Sypro Red. In comparison with covalently labeling proteins, the use of Sypro staining during electrophoretic separations not only presents a generic detection approach for the analysis of complex protein mixtures such as cell lysates but also avoids additional introduction of protein microheterogeneity as the result of labeling reaction. A comprehensive 2-D protein separation is completed in less than 10 min with an overall peak capacity of approximately 1700 using a chip with planar dimensions of as small as 2 cm x 3 cm. Significant enhancement in the peak capacity can be realized by simply raising the density of microchannels in the array, thereby increasing the number of IEF fractions further analyzed in the size-based separation dimension.     

Isobaric peptide termini labeling utilizing site-specific N-terminal succinylation

Recently, we introduced a novel approach for protein quantification based on isobaric peptide termini labeling (IPTL). In IPTL, both peptide termini are dervatized in two separate chemical reactions with complementary isotopically labeled reagents to generate isobaric peptide pairs. Here, we describe a novel procedure for the two chemical reactions to enable a cost-effective and rapid method. We established a selective N-terminal peptide modification reaction using succinic anhydride. Dimethylation was used as second chemical reaction to derivatize lysine residues. Both reactions can be performed within 15 min in one pot, and micropurification of the peptides between the two reactions was not necessary. For data analysis, we developed the force-find algorithm in IsobariQ which searches for corresponding peaks to build up peak pairs in tandem mass spectrometry (MS/MS) spectra where Mascot could not identify opposite sequences. Utilizing force-find, the number of quantified proteins was improved by more than 50% in comparison to the standard data analysis in IsobariQ. This was applied to compare the proteome of HeLa cells incubated with S-trityl-L-cysteine (STLC) to induce mitotic arrest and apoptosis. More than 50 proteins were found to be quantitatively changed, and most of them were previously reported in other proteome analyses of apoptotic cells. Furthermore, we showed that the two complementary isotopic labels coelute during liquid chromatography (LC) separation and that the linearity of relative IPTL quantification is not affected by a complex protein background. Combining the optimized reactions for IPTL with the open source data analysis software IsobariQ including force-find, we present a straightforward and rapid approach for quantitative proteomics.     

Analysis of gamma-carboxyglutamic acid content of protein, urine, and plasma by capillary electrophoresis and laser-induced fluorescence

When the properties of an analyte are known, the separation system can be designed to make the analyte of interest migrate at either a much faster or a much slower velocity compared to other molecules in the sample matrix. A simple and sensitive method to analyze the gamma-carboxyglutamic acid (Gla) content of protein, urine, and plasma was developed using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). The separation method is designed according to the specific properties of three amino acids of interest. The number of Gla residues from three vitamin K-dependent proteins were estimated by quantifying the amount of fluorescein thiocarbamyl derivative of Gla after alkaline hydrolysis and fluorescein isothiocyanate labeling. Human prothrombin, blood coagulation factor X, and bovine osteocalcin were calculated to have 10.0 +/- 0.7, 11.0 +/- 0.6, and 2.1 +/- 0.1 Gla residues per mole of protein, respectively, which agreed well with amino acid sequencing data. The analysis of free Gla content in urine and plasma was also demonstrated by this method. It was demonstrated that submicrograms of protein can be characterized by CE-LIF.     

Dynamic labeling during capillary or microchip electrophoresis for laser-induced fluorescence detection of protein-SDS complexes without pre- or postcolumn labeling.

The analysis of proteins under denaturing conditions is routinely performed with SDS-polyacrylamide gel electrophoresis. The automated capabilities of CE, use of nongel sieving matrixes, and on-line optical detection by either ultraviolet (UV) absorption or laser-induced fluorescence (LF) promise to revolutionize this method. While direct on-line detection of proteins is possible as a result of their intrinsic ability to absorb light in the UV part of the spectrum (detection sensitivity comparable to Coomassie Blue staining of gels), LIF provides more powerful detection but requires pre- or postcolumn fluorescence labeling of the proteins. The development of a protocol analogous to that used for double-stranded DNA analysis, where fluorescent intercalating dyes are simply included in the separation medium, would simplify size-based protein analysis immensely. This would avoid the complications associated with covalent modification of the proteins but still exploit the sensitivity of LIF detection. We demonstrate that this is possible with CE and microchip detection by incorporating, into the run buffer, a fluorescent dye that interacts hydrophobically with protein-SDS complexes. Key to this is a dye that fluoresces significantly when bound to protein-SDS complexes but not when bound to SDS micelles. Comparison of electropherograms from CE-based denaturing protein analysis with UV and LIF detection indicates that the presence of the fluor does not alter separation of the proteins. Moreover, comparison with electropherograms generated from microchip electrophoresis with LIF detection shows that equivalent patterns can be obtained. Despite the unoptimized nature of this separation system, a dynamic labeling protocol that allows for LIF detection for proteins is attractive and has the potential to circumvent the tedious labeling steps typically required.     

C-terminal sequence analysis of peptides and proteins using carboxypeptidases and mass spectrometry after derivatization of Lys and Cys residues

C-Terminal sequence analysis of peptides and proteins using carboxypeptidase digestion in combination with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is convenient for protein and peptide characterization. After a short digestion, a sequence up to 20 residues can be identified, but the total number depends on the individual sequence. Due to the accuracy limits of the MALDI time-of-flight arrangement, the assignment of several residues with close mass values, including Lys/Glx, may remain ambiguous. We have used derivatization of lysine residues by guanidination to overcome the problem of Lys identification. The reaction is rapid and specific and results in full derivatization. In the case of Cys-containing peptides, problems arise from the fact that carboxypeptidases Y and P do not cleave peptides that contain nonderivatized cystine, cysteic acid, or (carboxymethyl)cysteine. Successful identification of Cys residues within the sequence is instead achieved by conversion of Cys to 4-thialaminine by (trimethylamino)-ethylation. The two derivatizations of Lys and Cys side chains provide opportunities for proton attachment and therefore facilitate the analysis by MALDI-MS. This C-terminal sequence analysis method is also useful for large proteins after fragmentation with specific enzymes.     

Complications in the assignment of 14 and 28 Da mass shift detected by mass spectrometry as in vivo methylation from endogenous proteins

Identification of protein methylation sites typically starts with database searching of MS/MS spectra of proteolytic digest of the target protein by allowing addition of 14 and 28 Da in the selected amino acid residues that can be methylated. Despite the progress in our understanding of lysine and arginine methylation, substrates and functions of protein methylation at other amino acid residues remain unknown. Here we report the analysis of protein methylation for p53, SMC3, iNOS, and MeCP2. We found that a large number of peptides can be modified on the lysine, arginine, histidine, and glutamic acid residues with a mass increase of 14 or 28 Da, consistent with methylation. Surprisingly, a majority of which did not demonstrate a corresponding mass shift when cells were cultured with isotope-labeled methionine, a precursor for the synthesis of S-adenosyl-l-methionine (SAM), which is the most commonly used methyl donor for protein methylation. These results suggest the possibility of either exogenous protein methylation during sample handling and processing for mass spectrometry or the existence of SAM-independent pathways for protein methylation. Our study found a high occurrence of protein methylation from SDS-PAGE isolated endogenous proteins and identified complications for assigning such modifications as in vivo methylation. This study provides a cautionary note for solely relying on mass shift for mass spectrometric identification of protein methylation and highlights the importance of in vivo isotope labeling as a necessary validation method.     

Electrophoretic separation of proteins on a microchip with noncovalent, postcolumn labeling

Proteins were separated by microchip capillary electrophoresis and labeled on-chip by postcolumn addition of a fluorogenic dye, NanoOrange, for detection by laser-induced fluorescence. NanoOrange binds noncovalently with hydrophobic protein regions to form highly fluorescent complexes. Kinetic measurements of complex formation on the microchips suggest that the reaction rate is near the diffusion limit under the conditions used for protein separation. Little or no band broadening is caused by the postcolumn labeling step. Lower limits of detection for model proteins, alpha-lactalbumin, beta-lactoglobulin A, and beta-lactoglobulin B, were <0.5 pg (approximately 30 amol) of injected sample. The relative fluorescence and reaction rates are compared with those of a number of other fluorogenic dyes used for protein labeling.     

Revealing a positive charge patch on a recombinant monoclonal antibody by chemical labeling and mass spectrometry

During purification process development and analytical characterization, a recombinant human monoclonal antibody, referred to as rmAb1, showed an anomalous charge heterogeneity profile by cation-exchange chromatography (CIEC), characterized by extremely high retention and poor resolution between charge variants. Mass spectrometry-based footprinting methodologies that include selective labeling of lysine with sulfosuccinimidyl acetate and arginie with p-hydroxyphenylglyoxal were developed to map the positive charges on the rmAb1 surface. On the basis of the average percentages of labeling obtained for the lysine and arginine residues by peptide mapping analysis, the positive charges were more distributed on the surface in the Fab region than in the Fc region of rmAb1. By a comparative study of in-solution and on-resin labeling reaction dynamics, seven positively charged residues were identified to bind to the cation-exchange resin and they were located in the variable domains. Among them, three lysine and one arginine residues appeared to cluster together on the surface to form a positive charge patch. When the charge patch residues were neutralized by chemical labeling, rmAb1 exhibited a more typical CIEC retention time, confirming that the charge patch was responsible for the atypical CIEC profile of rmAb1. To our knowledge, this work is the first report revealing the amino acid composition of a surface charge patch on therapeutic monoclonal antibodies.     

Partial acetylation of lysine residues improves intraprotein cross-linking

Intramolecular cross-linking coupled with mass spectrometric identification of cross-linked amino acids is a rapid method for elucidating low-resolution protein tertiary structures or fold families. However, previous cross-linking studies on model proteins, such as cytochrome c and ribonuclease A, identified a limited number of peptide cross-links that are biased toward only a few of the potentially reactive lysine residues. Here, we report an approach to improve the diversity of intramolecular protein cross-linking starting with a systematic quantitation of the reactivity of lysine residues of a model protein, bovine cytochrome c. Relative lysine reactivities among the 18 lysine residues of cytochrome c were determined by the ratio of d0 and acetyl-d3 groups at each lysine after partial acetylation with sulfosuccinimidyl acetate followed by denaturation and quantitative acetylation of remaining unmodified lysines with acetic-d6 anhydride. These lysine reactivities were then compared with theoretically derived pKa and relative solvent accessibility surface values. To ascertain if partial N-acetylation of the most reactive lysine residues prior to cross-linking can redirect and increase the observable Lys-Lys cross-links, partially acetylated bovine cytochrome c was cross-linked with the amine-specific, bis-functional reagent, bis(sulfosuccinimidyl)suberate. After proteolysis and mass spectrometry analysis, partial acetylation was shown to significantly increase the number of observable peptides containing Lys-Lys cross-links, shifting the pattern from the most reactive lysine residues to less reactive ones. More importantly, these additional cross-linked peptides contained novel Lys-Lys cross-link information not seen in the non-acetylated protein and provided additional distance constraints that were consistent with the crystal structure and facilitated the identification of the proper protein fold.     

Trypsin immobilization on hairy polymer chains hybrid magnetic nanoparticles for ultra fast, highly efficient proteome digestion, facile 18O labeling and absolute protein quantification

In recent years, quantitative proteomic research attracts great attention because of the urgent needs in biological and clinical research, such as biomarker discovery and verification. Currently, mass spectrometry (MS) based bottom up strategy has become the method of choice for proteomic quantification. In this strategy, the amount of proteins is determined by quantifying the corresponding proteolytic peptides of the proteins, therefore highly efficient and complete protein digestion is crucial for achieving accurate quantification results. However, the digestion efficiency and completeness obtained using conventional free protease digestion is not satisfactory for highly complex proteomic samples. In this work, we developed a new type of immobilized trypsin using hairy noncross-linked polymer chains hybrid magnetic nanoparticle as the matrix aiming at ultra fast, highly efficient proteomic digestion and facile (18)O labeling for absolution protein quantification. The hybrid nanoparticle is synthesized by in situ growth of hairy polymer chains from the magnetic nanoparticle surface using surface initiated atom transfer radical polymerization technique. The flexible noncross-linked polymer chains not only provide large amount of binding sites but also work as scaffolds to support three-dimensional trypsin immobilization which leads to increased loading amount and improved accessibility of the immobilized trypsin. For complex proteomic samples, obviously increased digestion efficiency and completeness was demonstrated by 27.2% and 40.8% increase in the number of identified proteins and peptides as well as remarkably reduced undigested proteins residues compared with that obtained using conventional free trypsin digestion. The successful application in absolute protein quantification of enolase from Thermoanaerobacter tengcongensis protein extracts using (18)O labeling and MRM strategy further demonstrated the potential of this hybrid nanoparticle immobilized trypsin for high throughput proteome quantification.     

Multiple labeling of proteins

Fluorescent dyes are often used to label proteins before analysis by capillary electrophoresis. Fluorescent labeling produces spectacular improvements in sensitivity compared with UV absorbance detection of the native protein. However, labeling of the protein can lead to significant band broadening. This band broadening is interpreted as a result of multiple labeling of the protein, wherein one or more fluorescent molecules are bound to the protein. The heterogeneous reaction products, which are presumed to have different mobilities, generate a broad peak during electrophoresis. There has been little direct evidence for multiple labeling as the cause of band broadening of proteins. In this paper, we perform electrophoresis on native green fluorescence protein, along with the reaction products produced by fluorescence labeling. For short incubations, a series of regularly spaced components are resolved by free-zone electrophoresis; upon longer incubation, the product peaks merge together, forming a broad envelope.     

Automated ultra-thin-layer SDS gel electrophoresis of proteins using noncovalent fluorescent labeling

Ultra-thin-layer SDS gel electrophoresis in conjunction with automated laser-induced fluorescence detection is a novel and powerful method for the analysis of fluorophore-labeled proteins. The technique described in this paper employs instant, noncovalent fluorophore labeling by the addition of a fluorescent staining dye to the sample proteins either during or immediately prior to the sample loading process. Thus, the method does not require time-consuming post- or preseparation staining/labeling. By combining the multilane format of SDS polyacrylamide slab gel electrophoresis and the high separation efficiency of capillary SDS gel electrophoresis, ultra-thin-layer SDS gel electrophoresis features rapid, high-throughput, and high-resolution analysis of proteins in the molecular mass range of 14-116 kDa. The good heat dissipation inherent to the ultrathin format enables the use of agarose and agarose-based composite separation matrixes, which can be easily replaced within the separation platform. Labeling efficiency as a function of the concentration of the staining dye, SDS, and proteins is thoroughly discussed. Detection sensitivity of the method was found to be at the low-femtomole level (1.25 ng/band), determined by analyzing a set of serial dilutions of standard proteins. Practical example of molecular mass determination and characterization of a complex protein mixture are also shown.     

Glycoprotein characterization combining intact protein and glycan analysis by capillary electrophoresis-electrospray ionization-mass spectrometry

Glycosylated proteins play important roles in a large number of biological processes. Therefore, a complete characterization in terms of glycan structures and glycoform heterogeneity is needed. In this paper, a combined approach based on glycan and intact glycoprotein analysis by capillary zone electrophoresis-electrospray-mass spectrometry (CZE-ESI-MS) is presented. Based on a new capillary coating, a CZE-ESI-MS method for the separation and characterization of intact glycoproteins has been developed and compared to a method recently introduced for the characterization of erythropoietin. The excellent glycoform separation results in high-quality mass spectra, high dynamic range, and good sensitivity, allowing the correct characterization of minor glycan modifications. Additionally, a CZE-ESI-MS separation method for underivatized N-glycans has been developed. The separation of glycans differing in the degree of sialic acids and repeats of noncharged carbohydrates is achieved. The separation power of the method is demonstrated by obtaining mobility differences in glycans differing only by 16 Da. A time-of-flight mass spectrometer allowed the correct identification of the glycan composition based on high mass accuracy and resolution, identifying even minor modifications such as the exchange of "O" by "NH". An ion trap mass spectrometer provided structural information of the underivatized glycans from fragmentation spectra. The general applicability of both methods to glycoprotein analysis is illustrated for erythropoietin, fetuin, and alpha1-acid glycoprotein. The results obtained by the glycan analysis allowed an unequivocal glyco-assignment to the masses obtained for the intact proteins as long as the protein backbone is well characterized. Furthermore, modifications found for intact proteins can be attributed to differences in the glycostructure.     

Protein labeling enhances aptamer selection by methods of kinetic capillary electrophoresis

Methods of kinetic capillary electrophoresis (KCE) facilitate highly efficient selection of DNA aptamers for protein targets. The inability to detect native proteins at low concentrations in capillary electrophoresis creates, however, a significant obstacle for many important protein targets. Here we suggest that protein labeling with new Chromeo dyes can help to overcome this obstacle. By labeling a number of proteins with Chromeo P503, we show that the labeling procedure enables accurate detection of proteins in CE without significantly affecting their electrophoretic mobility or their ability to bind DNA. Moreover, Chromeo P503 does not appear to label the amino-groups of buffer components to a significant extent, making the labeling procedure compatible with a large number of selection and run buffers. Fluorescent labeling of protein targets with Chromeo dyes empowers selection of aptamers by KCE methods and promises to increase the rate at which aptamers for new targets are being developed and introduced in various applications.     

High-throughput global peptide proteomic analysis by combining stable isotope amino acid labeling and data-dependent multiplexed-MS/MS

In this work, we describe the application of a stable isotope amino acid (lysine) labeling in conjunction with data-dependent multiplexed tandem mass spectrometry (MS/MS) to facilitate the characterization and identification of peptides from proteomic (global protein) digests. Lysine auxotrophic yeast was grown in the presence of 13C-labeled or unlabeled lysine and combined after harvesting in equal proportions. Endoproteinase LysC digestion of the cytosolic fraction produced a global proteomic sample, consisting of heavy/light labeled peptide pairs. Then data-dependent multiplexed-MS/MS was applied to simultaneously select and dissociate only labeled peptide ion pairs. The approach allows differentiation between N-terminal (e.g., b-type ions) and C-terminal fragment ions (e.g., y-type ions) in resulting tandem mass spectra, as well as the capability of differentiation between near-isobaric glutamine and lysine residues. We also describe the utility of peptide composition and fragment information to support peptide identifications and examine the potential application of lysine labeling for differential quantitative protein analysis.     

Integrated microfluidic system enabling protein digestion, peptide separation, and protein identification

An integrated platform is presented for rapid and sensitive protein identification by on-line protein digestion and analysis of digested proteins using electrospray ionization mass spectrometry or transient capillary isotachophoresis/capillary zone electrophoresis with mass spectrometry detection. A miniaturized membrane reactor is constructed by fabricating the microfluidic channels on a poly(dimethylsiloxane) substrate and coupling the microfluidics to a poly(vinylidene fluoride) porous membrane with the adsorbed trypsin. On the basis of he large surface area-to-volume ratio of porous membrane media, adsorbed trypsin onto the poly(vinylidene fluoride) membrane is employed for achieving ultrahigh catalytic turnover. The extent of protein digestion in a miniaturized membrane reactor can be directly controlled by the residence time of protein analytes inside the trypsin-adsorbed membrane, the reaction temperature, and the protein concentration. The resulting peptide mixtures can either be directly analyzed using electrospray ionization mass spectrometry or further concentrated and resolved by electrophoretic separations prior to the mass spectrometric analysis. This microfluidic system enables rapid identification of proteins in minutes instead of hours, consumes very little sample (nanogram or less), and provides on-line interface with upstream protein separation schemes for the analysis of complex protein mixtures such as cell lysates.     

Improving aptamer selection efficiency through volume dilution, magnetic concentration, and continuous washing in microfluidic channels

The generation of nucleic acid aptamers with high affinity typically entails a time-consuming, iterative process of binding, separation, and amplification. It would therefore be beneficial to develop an efficient selection strategy that can generate these high-quality aptamers rapidly, economically, and reproducibly. Toward this goal, we have developed a method that efficiently generates DNA aptamers with slow off-rates. This methodology, called VDC-MSELEX, pairs the volume dilution challenge process with microfluidic separation for magnetic bead-assisted aptamer selection. This method offers improved aptamer selection efficiencies through the application of highly stringent selection conditions: it retrieves a small number (<10(6)) of magnetic beads suspended in a large volume (>50 mL) and concentrates them into a microfluidic chamber (8 μL) with minimal loss for continuous washing. We performed three rounds of the VDC-MSELEX using streptavidin (SA) as the target and obtained new DNA aptamer sequences with low nanomolar affinity that specifically bind to the SA proteins.     

Pitfalls in protein quantitation using acid-catalyzed O18 labeling: hydrolysis-driven deamidation

Proteolysis combined with O(18) labeling emerged recently as a powerful tool for quantitation of proteins for which suitable internal standards cannot be produced using molecular biology methods. Several recent reports suggested that acid-catalyzed O(18) labeling may be superior to the commonly accepted enzymatic protocol, as it may allow more significant spacing between the isotopic clusters of labeled and unlabeled peptides, thereby eliminating signal interference and enhancing the quality of quantitation. However, careful examination of this procedure reveals that the results of protein quantitation assisted by acid-catalyzed O(18) labeling are highly peptide-dependent. The inconsistency was found to be caused by deamidation of Asn, Gln, and carbamidomethylated Cys residues during prolonged exposure of the proteolytic fragments to the acidic environment of the labeling reaction, which translates into a loss in signal for these peptides. Taking deamidation into account leads to a significant improvement in the consistency of quantitation across a range of different proteolytic fragments.