Top down characterization of larger proteins (45 kDa) by electron capture dissociation mass spectrometry.

The structural characterization of proteins expressed from the genome is a major problem in proteomics. The solution to this problem requires the separation of the protein of interest from a complex mixture, the identification of its DNA-predicted sequence, and the characterization of sequencing errors and posttranslational modifications. For this, the "top down" mass spectrometry (MS) approach, extended by the greatly increased protein fragmentation from electron capture dissociation (ECD), has been applied to characterize proteins involved in the biosynthesis of thiamin, Coenzyme A, and the hydroxylation of proline residues in proteins. With Fourier transform (FT) MS, electrospray ionization (ESI) of a complex mixture from an E. coli cell extract gave 102 accurate molecular weight values (2-30 kDa), but none corresponding to the predicted masses of the four desired enzymes for thiamin biosynthesis (GoxB, ThiS, ThiG, and ThiF). MS/MS of one ion species (representing approximately 1% of the mixture) identified it with the DNA-predicted sequence of ThiS, although the predicted and measured molecular weights were different. Further purification yielded a 2-component mixture whose ECD spectrum characterized both proteins simultaneously as ThiS and ThiG, showing an additional N-terminal Met on the 8 kDa ThiS and removal of an N-terminal Met and Ser from the 27 kDa ThiG. For a second system, the molecular weight of the 45 kDa phosphopantothenoylcysteine synthetase/decarboxylase (CoaBC), an enzyme involved in Coenzyme A biosynthesis, was 131 Da lower than that of the DNA prediction; the ECD spectrum showed that this is due to the removal of the N-terminal Met. For a third system, viral prolyl 4-hydroxylase (26 kDa), ECD showed that multiple molecular ions (+98, +178, etc.) are due to phosphate noncovalent adducts, and MS/MS pinpointed the overall mass discrepancy of 135 Da to removal of the initiation Met (131 Da) and to formation of disulfide bonds (2 x 2 Da) at C32-C49 and C143-C147, although 10 S-S positions were possible. In contrast, "bottom up" proteolysis characterization of the CoaBC and the P4H proteins was relatively unsuccessful. The addition of ECD substantially increases the capabilities of top down FTMS for the detailed structural characterization of large proteins.     

Separation and identification of photosynthetic antenna membrane proteins by high- performance liquid chromatography electrospray ionization mass spectrometry

Functional proteomics of membrane proteins is an important tool for the understanding of protein networks in biological membranes. Nevertheless, structural studies on this part of the proteome are limited. The present review attempts to cover the vast array of methods that have appeared in the last few years for separation and identification of photosynthetic proteins of thylakoid membranes present in chloroplasts, a good model for setting up analytical methods suitable for membrane proteins. The two major methods for the separation of thylakoid membrane proteins are gel electrophoresis and liquid chromatography. Isoelectric focusing in a first dimension followed by denaturing sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) in a second dimension is an effective way to resolve large numbers of soluble and peripheral membrane proteins. However, it is not applicable for isolation of native protein complexes or for the separation of highly hydrophobic membrane proteins. High-performance liquid chromatography (HPLC), on the other hand, is highly suitable for any type of membrane protein separation due to its compatibility with detergents that are necessary to keep the hydrophobic proteins in solution. With regard to the identification of the separated proteins, several methods are available, including immunological and mass spectrometric methods. Besides immunological identification, peptide mass fingerprinting, peptide fragment fingerprinting or intact molecular mass determination by electrospray ionization mass spectrometry (ESI-MS) or matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) have been shown to be very sensitive and effective. In particular, identification of proteins by their intact molecular mass is advantageous for the investigation of numerous biological problems, because it is rapid and reflects the full sequence of the protein and all its posttranslational modifications. However, intact molecular mass determinations of gel-separated membrane proteins are hampered due to the difficulties in extracting the hydrophobic proteins from the gel, whereas HPLC on-line interfaced with ESI-MS enables the rapid and accurate determination of intact molecular masses and consequently an unequivocal protein identification. This strategy can be viewed as a multidimensional separation technique distinguishing between hydrophobicity in the first dimension and between different mass-to-charge ratios in the second dimension, allowing the separation and identification even of isomeric forms.     

Mass spectrometric analysis of porcine rhodopsin

Rhodopsin is the dim light photosensitive pigment of animals. In this work, we undertook to study the structure of rhodopsin from swine and compare it with bovine and rat rhodopsin. Porcine rhodopsin was analyzed using methodology developed previously for mass spectrometric analysis of integral membrane proteins. Combining efficient protein cleavage and high performance liquid chromatography separation with the sensitivity of mass spectrometry (MS), this technique allows the observation of the full protein map and the posttranslational modifications of the protein in a single experiment. The rhodopsin protein from a single porcine eye was sequenced completely, with the exception of two single-amino acid fragments and one two-amino acid fragment, and the gene sequence reported previously was confirmed. The posttranslational modifications, similar to the ones reported previously for bovine and rat rhodopsin, were also identified. Although porcine rhodopsin has a high degree of homology to bovine and rat rhodopsins and most of their posttranslational modifications are identical, the glycosylation and phosphorylation patterns observed were different. These results show that rhodopsin from a single porcine eye can be characterized completely by MS. This technology opens the possibility of rhodopsin structural and functional studies aided by powerful mass spectrometric analysis, using the fellow eye as an internal control.     

Analysis of the saliva from patients with oral cancer by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), this study analyzed the saliva obtained from patients with oral cancer and compared these mass spectra with those obtained from healthy controls. Saliva without pre-treatment was mixed directly with a sinapinic acid matrix. Alpha-amylase (57 kDa) dominated the high mass range in the MALDI mass spectra of the saliva from healthy subjects, but the peak was suppressed for patients with oral cancer and was replaced by a peak at m/z 66 k in the spectra of patients' samples (15 out of 20). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with in-gel tryptic digestion combined with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) was employed to characterize this 66-kDa protein, which was thus shown to be albumin. However, based on SDS-PAGE results, concentrations of both alpha-amylase and albumin in patients' saliva were significantly higher than those in healthy subjects. This discrepancy was shown to be due to MALDI suppression effects due to the albumin. MALDI-MS thus has potential as a possible rapid diagnostic screening tool for oral cancer.     

Protein profiles of the Chinese hamster ovary cells in the resting and proliferating stages.

Identification and characterization of the proteins that regulate the transition from the resting stage (G0) through G1 to S phase of the cell cycle are of central importance to understand the control of cell proliferation and chromosome replication. Unlike in lower organisms, where relatively small numbers of key factors are involved in this process, the factors involved in the same control mechanisms in mammalian systems are much more complex. Furthermore, accumulating lines of evidence now suggest that the nuclear matrix and chromatin organization also play an essential role for the cell cycle control in mammalian cells. To gain a better understanding of the overall dynamics and changes of the protein factors in the context of matrix/chromatin organization, we examined the protein profiles of the Chinese hamster ovary (CHO) cells in different cell cycle compartments. The methods used in this study included subcellular fractionations (cytosol, nuclear extraction, chromatin, and nuclear matrix), two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), silver staining, and immunoblotting. As expected, significant changes of protein profiles were observed when cells entered into proliferating stages from G0. Among approximately 1200 protein spots analyzed by 2-D PAGE, at least 12 showed marked increase or decrease at this transitional period. Further cell-cycle progression from G1 to S phase showed less dramatic changes of overall protein protile. However, the profile of certain proteins showed rather dramatic changes of their subcellular localization during this transitional period. In particular, the levels of proliferating cell nuclear antigen (PCNA) in the nuclear matrix and chromatin dramatically increased in mid-G1 and in the beginning of S phase, respectively, while the overall PCNA level was relatively constant throughout the cell cycle.     

Effects of ultraviolet B exposure on the expression of proliferating cell nuclear antigen in murine skin

Proliferating cell nuclear antigen (PCNA) is an active nuclear protein involved in DNA replication, recombination and repair. PCNA is found throughout the basal layer in normal skin and in all layers of the epidermis in malignancy. This study evaluates PCNA's expression after acute and chronic UV-B irradiation. Skh-1 hairless mice exposed to 1.5 and 4.5 kJ/m2 of UV-B were sacrificed at 6, 12, 24, 48, 72 and 168 h. Immunohistochemical analysis revealed PCNA expression throughout the basal layer of untreated skin, with diminished expression at 6 h, indicative of immediate UV damage, and evidenced by the observable upregulation in pyrimidine dimer formation early on. Subsequently, PCNA immunoreactivity progressively increased, demonstrating an aberrant upward epidermal migratory pattern in association with chronic exposure. The 4.5 kJ/m2 group exhibited prolonged recovery in staining and also demonstrated this altered migratory pattern with chronic exposure. Progressive reactivation of PCNA expression occurs with repair. PCNA migration to upper layers of the epidermis indicates proliferation and possibly a subsequent increased malignant potential. We conclude that PCNA can serve as a marker of DNA repair and indirectly as an indicator of UV-B-induced damage, expression being time dependent and dose related. Specific immunoreactivity patterns and the observable atypia in keratinocytes are relevant in elucidating malignant potentiation.     

A comparative study of CE-SDS,SDS-PAGE,and Simple Western: Influences of sample preparation on molecular weight determination of proteins

The development of capillary electrophoresis, especially CE-SDS devices, has led CE-SDS to become an established tool in a wide range of applications in the analysis of biopharmaceuticals and is increasingly replacing its method of origin, SDS-PAGE. The goal of this study was to evaluate the comparability of molecular weight (MW) determination especially by CE-SDS and SDS-PAGE. For ensuring comparability, model proteins that have little or no posttranslational modifications and an IgG antibody were used. Only a minor influence of sample preparation conditions, including sample buffer, temperature conditions, and different reducing agents on the MW determination were found. In contrast, the selection of the MW marker plays a decisive role in determining the accurate apparent MW of a protein. When using different MW markers, the deviation in MW determination can exceed 10%. Interestingly, CE-SDS and 10% SDS-PAGE hardly differ in their trueness of MW determination. The trueness in relation to the reference MW for each protein was calculated. Although the trueness values for the model proteins considered range between 1.00 and 1.11 using CE-SDS, they range between 0.93 and 1.03 on SDS-PAGE, depending on the experimental conditions chosen.     

Arginylation and methylation double up to regulate nuclear proteins and nuclear architecture in vivo

Protein arginylation and arginine methylation are two posttranslational modifications of emerging importance that involve Arg residues and their modifications. To test a hypothesis that posttranslationally added arginines can be methylated, we used high-precision mass spectrometry and metabolic labeling to find whether posttranslationally added arginines can serve as methylation sites. We identified?a number of proteins in?vivo, on which posttranslationally added Arg have undergone mono- and dimethylation. This double modification predominantly affects the chromatin-containing nuclear fraction and likely plays an important regulatory role in chromatin-associated proteins. Moreover, inhibition of arginylation and Arg methylation results in?a significant reduction of the nucleus size in cultured cells, suggesting changes in chromatin compaction and nuclear architecture. Our findings suggest?a functional link between protein regulation by arginylation and methylation that affects nuclear structure in?vivo.     

Immobilization of a Bacterial Cytochrome P450 Monooxygenase System on a Solid Support

Bacterial cytochrome P450s (P450s), which catalyze regio‐ and stereoselective oxidations of hydrocarbons with high turnover rates, are attractive biocatalysts for fine chemical production. Enzyme immobilization is needed for cost‐effective industrial manufacturing. However, immobilization of P450s is difficult because electron‐transfer proteins are involved in catalysis and anchoring these can prevent them from functioning as shuttle molecules for carrying electrons. We studied a heterotrimeric protein‐mediated co‐immobilization of a bacterial P450, and its electron‐transfer protein and reductase. Fusion with subunits of a heterotrimeric Sulfolobus solfataricus proliferating cell nuclear antigen (PCNA) enabled immobilization of the three proteins on a solid support. The co‐immobilized enzymes catalyzed monooxygenation because the electron‐transfer protein fused to PCNA via a single peptide linker retained its electron‐transport function.     

Total synthesis of TRADD death domain with arginine N-GlcNAcylation by hydrazide-based native chemical ligation

TNFR1-associated death domain protein(TRADD)with arginine N-GlcNAcylation is a novel and structurally unique posttranslational modification(PTM)glycoprotein that blocks the formation of death-inducing signaling complex(DISC),orchestrating host nuclear factorκB(NF-κB)signaling in entero-pathogenic Escherichia coli(EPEC)-infected cells.This particular glycosylated modification plays an extremely vital role for the effective colonization and pathogenesis of pathogens in the gut.Herein we describe the total synthesis of TRADD death domain(residues 195-312)with arginine235 NGlcNAcylation(Arg-GIcNAc TRADD(195-312)).Two longish peptidyl fragments of the wild-type primary sequence were obtained by robust,microwave-assisted,highly efficient,solid-phase peptide synthesis(SPPS),the N-GlcNAcylated sector was built by total synthesis and attached specifically to resinbound peptide with an unprotected ornithine residue via silver-promoted on-resin guanidinylation,ArgGlcNAc TRADD(195-312)was constructed by hydrazide-based native chemical ligation(NCL).The facile synthetic strategy is expected to be generally applicable for the rapid synthesis of other proteins with Arg-GIcNAc modification and to pave the way for the related chemically biological study.     

Posttranslational modifications and subcellular localization signals: indicators of sequence regions without inherent 3D structure?

Given the huge number of sequences of otherwise uncharacterized protein sequences, computer-aided prediction of posttranslational modifications (PTMs) and translocation signals from amino acid sequence becomes a necessity. We have contributed to this multi-faceted, worldwide effort with the development of predictors for GPI lipid anchor sites, for N-terminal N-myristoylation sites, for farnesyl and geranylgeranyl anchor attachment as well as for the PTS1 peroxisomal signal. Although the substrate protein sequence signals for various PTMs or translocation systems vary dramatically, we found that their principal architecture is similar for all the cases studied. Typically, a small stretch of the amino acid residues is buried in the catalytic cleft of the protein-modifying enzyme (or the binding site of the transporter). This piece most intensely interacts with the enzyme and its sequence variability is most restricted. This stretch is surrounded by linker segments that connect the part bound by the enzyme with the rest of the substrate protein. These residues are, as a trend, small with a flexible backbone and polar. Due to the mechanistic requirements of binding to the enzyme, we suggest that most PTM sites are necessarily embedded into intrinsically disordered regions (except for cases of autocatalytic PTMs, PTMs executed in the unfolded state or non-enzymatic PTMs) and this issue requires consideration in structural studies of proteins with complex architecture. Surprisingly, some proteins carry sequence signals for posttranslational modification or translocation that remain hidden in the normal biological context but can become fully functional in certain conditions.     

Cancer proteomics: from identification of novel markers to creation of artifical learning models for tumor classification

Studies of global protein expression in human tumors have led to the identification of various polypeptide markers, potentially useful as diagnostic tools. Many changes in gene expression recorded between benign and malignant human tumors are due to post-translational modifications, not detected by analyses of RNA. Proteome analyses have also yielded information about tumor heterogeneity and the degree of relatedness between primary tumors and their metastases. Results from our own studies have shown a similar pattern of changes in protein expression in different epithelial tumors, such as decreases in tropomyosin and cytokeratin expression and increases in proliferating cell nuclear antigen (PCNA) and heat shock protein expression. Such information has been used to create artificial learning models for tumor classification. The artificial learning approach has potential to improve tumor diagnosis and cancer treatment prediction.     

Molecular mass determination of plasma-derived glycoproteins by ultraviolet matrix-assisted laser desorption/ionization time-of-flight mass spectrometry with internal calibration

Human plasma-derived antithrombin III (AT-III), factor IX (FIX) and vitronectin (VN) were characterized as native glycoproteins and in their de-N-glycosylated form by means of MALDI mass spectrometry. The average molecular masses of the three complex glycoproteins were determined applying internal calibration with high-mass, well-defined protein calibrants. Internal calibration generated for the 47 kDa yeast protein enolase a mass precision in the continuous and delayed extraction mode of +/-0.12 and +/-0.022%, respectively. The achievable mass accuracy for such a high-mass, unmodified protein was in the range of 0.02% in the continuous mode, which turned out to be better than in the delayed extraction mode. Purification of all (glyco) proteins (even the calibration proteins) by means of ZipTip technology and direct elution with a solvent system containing the appropriate MALDI matrix turned out to be a prerequisite to measure the exact molecular masses with an internal calibration. The average molecular masses of the two different forms of AT-III, namely AT-III(alpha) and AT-III(beta), were shown to be 57.26 and 55.04 kDa, respectively. The 2.22 kDa mass difference is attributed to the known difference in carbohydrate content at one specific site (Asn-135). After exhaustive de-N-glycosylation (by means of PNGase F) of the alpha- and beta-form and subsequent MALDI-MS analysis, average molecular masses of 48.96 and 48.97 kDa, respectively, were obtained. These values are in good agreement (-0.15%) with the calculated molecular mass (49.039 kDa) of the protein part based on SwissProt data. The molecular mass of the heavily post-translational modified glycoprotein FIX was found to be 53.75 kDa with a peak width at 10% peak height of 4.5 kDa, because of the presence of many different posttranslational modifications (N- and O-glycosylation at multiple sites, sulfation, phosphorylation, hydroxylation and numerous gamma-carboxyglutamic acids). MALDI-MS molecular mass determination of the native, size-exclusion chromatography-purified, VN sample revealed that the glycoprotein was present as dimer with molecular mass of 117.74 kDa, which could be corroborated by non-reducing SDS-PAGE. After sample treatment with guanidine hydrochloride and mass spectrometric analysis, a single, new main component was detected. The molecular mass turned out to be 59.45 kDa, representing the monomeric form of VN, known as V75. The determined molecular mass value was shown to be on one hand lower than from SDS-PAGE and on the other higher than the calculated amino acid sequence molecular mass (52 277 Da), pointing to the well-known SDS-PAGE bias and to considerable post-translational modifications. Further treatment of the sample with a reducing agent and subsequent MALDI-MS revealed two new components with molecular masses of 49.85 and 9.41 kDa, corresponding to V65 and V10 subunits of VN. PNGase F digest of the V75 and V65 units and MS analysis, exhibiting a molecular mass reduction of 6.37 kDa in both cases, verified the presence of a considerable amount of N-glycans.     

Eukaryotic clamp loaders and unloaders in the maintenance of genome stability

Eukaryotic sliding clamp proliferating cell nuclear antigen (PCNA) plays a critical role as a processivity factor for DNA polymerases and as a binding and acting platform for many proteins. The ring-shaped PCNA homotrimer and the DNA damage checkpoint clamp 9-1-1 are loaded onto DNA by clamp loaders. PCNA can be loaded by the pentameric replication factor C (RFC) complex and the CTF18-RFC-like complex (RLC) in vitro. In cells, each complex loads PCNA for different purposes; RFC-loaded PCNA is essential for DNA replication, while CTF18-RLC-loaded PCNA participates in cohesion establishment and checkpoint activation. After completing its tasks, PCNA is unloaded by ATAD5 (Elg1 in yeast)-RLC. The 9-1-1 clamp is loaded at DNA damage sites by RAD17 (Rad24 in yeast)-RLC. All five RFC complex components, but none of the three large subunits of RLC, CTF18, ATAD5, or RAD17, are essential for cell survival; however, deficiency of the three RLC proteins leads to genomic instability. In this review, we describe recent findings that contribute to the understanding of the basic roles of the RFC complex and RLCs and how genomic instability due to deficiency of the three RLCs is linked to the molecular and cellular activity of RLC, particularly focusing on ATAD5 (Elg1).Subject terms: DNA synthesis, Checkpoints     

Measurement of emulsion droplet sizes using PFG NMR and regularization methods

The droplet size distributions of emulsions have been measured using pulsed field gradient (PFG) nuclear magnetic resonance (NMR) for many years. This technique finds particular application with emulsions that are concentrated and/or opaque, since such emulsion systems are difficult to characterize by other methods. Most studies employing PFG techniques assume a lognormal form when extracting the droplet size distribution from the experimental data. It is clearly desirable to retrieve a droplet size distribution from the experimental data without assuming such a functional form. This is achieved for the first time using regularization techniques. Regularization based on the distribution area and on its second derivative are compared and assessed along with the following techniques for selecting the optimal regularization parameter: the L-curve method, generalized cross validation (GCV), and the discrepancy principle. Regularization is applied to both simulated data sets and experimental data. It is found that when the experimental error can be estimated accurately, the discrepancy principle with area regularization is the best approach. When the error is not known the GCV method, with second derivative regularization and allowing only nonnegative values, is most effective.     

Electrophoretic characterization of a novel cysteine protease produced by Vibrio harveyi

Electrophoretic characterization of a novel cysteine protease produced by pathogenic luminous Vibrio harveyi, originally isolated from diseased tiger prawn Penaeus monodon in Taiwan, is demonstrated in the present study using native polyacrylamide gel electrophoresis (native PAGE), sodium dodecyl sulfate-PAGE (SDS-PAGE), crossed immunoelectrophoresis (CIE) and isoelectric focusing (IEF) gels. The protease has a pI of 6.4 and exhibits a fast-migrating feature in native-PAGE and CIE gels indicating that it is a negatively charged protease. The protease electrophoresed as a 22 kDa protein band in native- and SDS-PAGE (in SDS - buffer with or without the presence of 2-mercaptoethanol) while it electrophoresed as a 38 kDa protein band in SDS-PAGE when the samples were boiled for 10 min prior to electrophoresis. The results reveal that the enzyme is an SDS-resistant monomeric protease and its high negative charge is not influenced by SDS (detergent) without boiling the sample. The present results are useful in determining proteins of similar nature to this unique cysteine protease.     

Determination of monoclonal antibody production in cell culture using novel microfluidic and traditional assays

This study compares microfluidic technology (Protein 200 LabChip Assay kit, Agilent 2100 Bioanalyzer, referred to here as Protein 200) to the traditional approach for protein analysis, one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), for the sizing and quantification of immunoglobulin G (IgG) in hybridoma cell cultures. Internal references differ between each method: purified IgG was used alone in SDS-PAGE while myosin (the upper marker) was added to each sample in Protein 200. The IgG used here were produced in cultures propagated in either a serum-free or a serum-containing medium. With serum-containing samples, there was a significant difference in the IgG concentrations (p < 0.05) between SDS-PAGE and Protein 200. The concentration determined by SDS-PAGE was significantly higher (> 30%) than by Protein 200 or by high-pressure liquid chromatography (HPLC) because the large amounts of serum albumin in the samples affect the accuracy of SDS-PAGE. Protein 200 can determine size similarly to SDS-PAGE in serum-free samples (standard error of the mean, SEM, < 1%, 95% confidence < +/-1%), unlike in serum-containing samples. The Protein 200 assay was more effective than the traditional one-dimensional SDS-PAGE in determining concentration and size of IgG in cell culture samples and it provided a miniaturized and convenient platform for rapid analysis.     

Establishing a quality control protocol for the radiochemical evaluation of radioiodinated biomolecules with application in nuclear medicine

Quality Control (QC) of radiopharmaceuticals is important, providing products of high standards. The current study refers to an enzymic radioiodination method applied to the labeling of antibodies used in nuclear medicine and to the respective QC procedures. Readily available materials such as¹²⁵I, polyclonal immunoglobulin G (IgG) and a collection of simple chromatographic techniques helped establish a QC protocol suitable for the radiochemical evaluation of radioiodinated biomolecules. A high pre-purification radiolabeling yield (90%) for total protein was detected by paper electrophoresis (PE) and size exclusion chromatography (SEC), but only the inclusion of SDS-PAGE confirmed the existence of “self-iodination” of lactoperoxidase (LPO) and revealed the true radiolabeling yield for¹²⁵I-IgG (79%).