Proteomic profiling of erythrocyte proteins by proteolytic digestion chip and identification using two-dimensional electrospray ionization tandem mass spectrometry

Self-assembled monolayers (SAMs) on coinage metal provide versatile modeling systems for studies of interfacial electron transfer, biological interactions, molecular recognition, and other interfacial phenomena. The bonding of enzyme to SAMs of alkanethiols onto gold surfaces is exploited to produce an enzyme chip. In this work, the attachment of trypsin to a SAMs surface of 11-mercaptoundecanoic acid was achieved using water soluble N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide as coupling agent. A two-dimensional liquid-phase separation scheme coupled with mass spectrometry is presented for proteomic analysis of erythrocyte proteins. The application of proteomics, particularly with reference to analysis of proteins, will be described. Surface analyses have revealed that the X-ray Photoelectron Spectroscopy (XPS) C1s and N1s core levels illustrate the immobilization of trypsin. These data are also in good agreement with Fourier Transformed Infrared Reflection-Attenuated Total Reflection (FTIR-ATR) spectra for the peaks at Amide I and Amide II. Using two-dimensional nano-high performance liquid chromatography electrospray ionization tandem mass spectrometry (2D nano-HPLC-ESI-MS/MS) system observations, analytical results have demonstrated the erythrocyte proteins digestion of the immobilized trypsin on the functionalized SAMs surface. For such surfaces, it also shows the enzyme digestion ability of the immobilized trypsin. The experiment results revealed the identification of 272 proteins from erythrocyte protein sample. The terminal groups of the SAMs structure can be further functionalized with biomolecules or antibodies to develop surface-base diagnostics, biosensors, or biomaterials.     

Sequencing oligonucleotides with blocked termini using exonuclease digestion and electrospray mass spectrometry

A method for sequencing ODNs with both termini blocked using mass spectrometry (MS) is reported. The ladder sequencing method is based on our investigation and understanding of critical factors affecting snake venom phosphodiesterase (SVP) digestion of such ODNs. To produce sequence ladders suitable for MS analysis, digestion conditions such as SVPs from three snake species and pH values of digestion buffer were investigated. SVP of Crotalus duressus terrificus (SVP I) was found to be the most suitable for sequencing ODNs with both termini blocked. The pH value of 9.4, which is optimal for SVP digestion of unmodified ODNs, was found to be unsuitable for ladder sequencing ODNs with both termini blocked. Instead, digestion in a wide range of pH values (pH 5-8), including rarely used acidic conditions, was found to be necessary to obtain otherwise unobtainable sequence information. With digestion buffer of desired pH values, sequence ladders which are recorded as MWs of truncated ODNs from SVP digestion are obtained. Examples of sequencing ODNs up to 26 bases long with both termini blocked are demonstrated in this work.     

Glycan profiling of monoclonal antibodies using zwitterionic-type hydrophilic interaction chromatography coupled with electrospray ionization mass spectrometry detection

We present a new method for the analysis of glycans enzymatically released from monoclonal antibodies (MAbs) employing a zwitterionic-type hydrophilic interaction chromatography (ZIC–HILIC) column coupled with electrospray ionization mass spectrometry (ESI–MS). Both native and reduced glycans were analyzed, and the developed procedure was compared with a standard HILIC procedure used in the pharmaceutical industry whereby fluorescent-labeled glycans are analyzed using a TSK Amide-80 column coupled with fluorescence detection. The separation of isobaric alditol oligosaccharides present in monoclonal antibodies and ribonuclease B is demonstrated, and ZIC–HILIC is shown to have good capability for structural recognition. Glycan profiles obtained with the ZIC–HILIC column and ESI–MS provided detailed information on MAb glycosylation, including identification of some less abundant glycan species, and are consistent with the profiles generated with the standard procedure. This new ZIC–HILIC method offers a simpler and faster approach for glycosylation analysis of therapeutic antibodies.     

Characterization of gel-separated glycoproteins using two-step proteolytic digestion combined with sequential microcolumns and mass spectrometry

Protein glycosylation can be vital for changing the function or physiochemical properties of a protein. Abnormal glycosylation can lead to protein malfunction, resulting in severe diseases. Therefore, it is important to develop techniques for characterization of such modifications in proteins at a sensitivity level comparable with state-of-the-art proteomics. Whereas techniques exist for characterization of high abundance glycoproteins, no single method is presently capable of providing information on both site occupancy and glycan structure on a single band excised from an electrophoretic gel. We present a new technique that allows characterization of low amounts of glycoproteins separated by gel electrophoresis. The method takes advantage of sequential specific and nonspecific enzymatic treatment followed by selective purification and characterization of the glycopeptides using graphite powder microcolumns in combination with mass spectrometry. The method is faster and more sensitive than previous approaches and is compatible with proteomic studies.     

Analysis of recombinant monoclonal antibody isoforms by electrospray ionization mass spectrometry as a strategy for streamlining characterization of recombinant monoclonal antibody charge heterogeneity

A therapeutic recombinant monoclonal antibody analyzed by cation-exchange chromatography exhibited a heterogeneous profile composed of approximately 10 isoforms. The peaks were isolated and characterized by electrospray quadrupole time-of-flight mass spectrometry (ESI-q-TOF-MS), N-terminal Edman sequencing, peptide mapping, and other techniques. Acidic (lower pI) peaks were found to represent deamidated and sialyated species. Higher pI peaks were found to contain N- and C-terminal heavy-chain variants. Biological activities of the more abundant isoforms were found to be comparable. An approach streamlining the characterization of antibody charge heterogeneity is proposed.     

Accurate mass analysis of phosphoramidites by electrospray mass spectrometry

A method of accurate mass determination of phosphoramidites is described. The commonly used methanol/water/acid system was replaced by LiCl-containing acetonitrile and the concentrations of LiCl, poly(ethylene glycol), and phosphoramidite samples were optimized.     

Protein analysis by electrospray ionization mass spectrometry

The applicability of electrospray ionization (ESI) mass spectrometry to protein analyses has been studied. The molecular weight of hen egg lysozyme (HEL) was determined with an accuracy of +/- 2 u. The choice of solvents and additives in sample preparations was important to achieve high sensitivity as well as high precision of molecular weight measurements.     

Rapid Fc glycosylation analysis of Fc fusions with IdeS and liquid chromatography mass spectrometry

We developed a rapid method to analyze Fc glycosylation of Fc fusion proteins, especially those with mutated Fc hinge regions. Fc fusion proteins were digested with IdeS, an IgG specific protease with exosites for substrate recognition and cleavage. The resultant fragments were directly analyzed through liquid chromatography mass spectrometry. The structures and relative quantities of Fc glycans were deduced from their masses and intensities. The separated substrate recognition and cleavage property of IdeS makes this method applicable to a broad range of Fc fusion proteins having either standard or non-canonical hinge regions.     

Kinetic analysis of NodST sulfotransferase using an electrospray ionization mass spectrometry assay

A novel and efficient enzyme kinetics assay using electrospray ionization mass spectrometry was developed and applied to the bacterial carbohydrate sulfotransferase (NodST). NodST catalyzes the sulfuryl group transfer from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to chitobiose, generating 3'-phosphoadenosine 5'-phosphate (PAP) and chitobiose-6-OSO(3)(-) as products. Traditional spectrophotometric assays are not applicable to the NodST system since no shift in absorption accompanies sulfuryl group transfer. Alternative assays have employed thin-layer chromatography, but this procedure is time-consuming and requires radioactive materials. The ESI-MS assay presented herein requires no chromophoric substrate or product, and the analysis time is very short. The ESI-MS assay is used to determine NodST kinetic parameters, including K(M), V(max), and K(i) (for PAP). In addition, the mode of inhibition for PAP was rapidly determined. The results were in excellent agreement with those obtained from previous assays, verifying the accuracy and reliability of the ESI-MS assay. This unique technique is currently being used to investigate the enzymatic mechanism of NodST and to identify sulfotransferase inhibitors.     

Analysis of hyaluronan content in chondroitin sulfate preparations by using selective enzymatic digestion and electrospray ionization mass spectrometry

Two important glycosaminoglycans (GAGs) with structural roles in the body's cartilage are hyaluronan (HA) and chondroitin sulfate (CS). A simple mass spectrometric method for determining the amount of HA that may be present in isolated CS samples is presented in this article. Samples are subjected to selective enzymatic digestion using a bacterial hyaluronidase (HA lyase, EC 4.2.2, from Streptococcus dysgalactiae) specific for HA. Undigested CS GAG is then removed by centrifugal filtration, and digested HA left in the filtrate is quantified by electrospray ionization mass spectrometry using an internal standard and selected ion monitoring. The described method was applied to the analysis of several CS samples prepared for use in nutritional supplements.     

Characterisation of glucosinolates using electrospray ion trap and electrospray quadrupole time-of-flight mass spectrometry

Twelve naturally occurring glucosinolates displaying alkenyl, hydroxylated, methylsulphinyl, aromatic and indole side chains were investigated by both negative and positive ion electrospray ionisation-tandem mass spectrometry (ESI-MS/MS). In order to resolve the MS/MS spectra obtained from the anion and cation molecular ions of glucosinolates, the different fragments were investigated by MSn experiments using an ion trap spectrometer. The MS3 spectra obtained permitted possible fragmentation schemes to be proposed. These were supported by accurate mass measurements of some characteristic diagnostic ions with the help of a quadrupole time-of-flight instrument. The negative ion ESI-MS/MS behaviour of the different glucosinolates investigated in this study confirmed previously described patterns and revealed new interesting structural informative fragments. Some are common to all the glucosinolates and others are highly specific for a type of variable side chain. The positive ion ESI-MS/MS fragments obtained from the [MNa+Na]+ or [MK+K]+ molecular ions did not provide complementary specific diagnostic ions. Nevertheless, when compared with the negative ion mode, the daughter ions appeared more homogenous and with a better relative abundance for all of the 12 compounds studied. Moreover, the positive ion mode appeared to be more efficient than the negative mode for the study of methoxylated glucosinolates and should be useful to detect the glucosinolates present as organic salts in crude plant extracts.     

Detection and quantification of free sulfhydryls in monoclonal antibodies using maleimide labeling and mass spectrometry

The detection of free sulfhydryls in proteins can reveal incomplete disulfide bond formation, indicate cysteine residues available for conjugation, and offer insights into protein stability and structure. Traditional spectroscopic methods of free sulfhydryl detection, such as Ellman’s reagent, generally require a relatively large amount of sample, preventing their use for the analysis of biotherapeutics early in the development cycle. These spectroscopic methods also cannot accurately determine the location of the free sulfhydryl, further limiting their utility. Mass spectrometry was used to detect free sulfhydryl residues in intact proteins after labeling with Maleimide-PEG₂-Biotin. As little as 2% cysteine residues with free sulfhydryls (0.02 mol SH per mol protein) could be detected by this method. Following reduction, the free sulfhydryl abundance on antibody heavy and light chains could be measured. To determine free sulfhydryl location at peptide-level resolution, free sulfhydryls and cysteines involved in disulfide bonds were differentially labeled with N-ethylmaleimide and d₅-N-ethylmaleimide, respectively. Following enzymatic digestion and nanoLC-MS, the abundance of free sulfhydryls at individual cysteine residues was quantified down to 2%. The method was optimized to avoid non-specific labeling, disulfide bond scrambling, and maleimide exchange and hydrolysis. This new workflow for free sulfhydryl analysis was used to measure the abundance and location of free sulfhydryls in 3 commercially available monoclonal antibody standards (NIST Monoclonal Antibody Reference Material (NIST), SILu?Lite SigmaMAb Universal Antibody Standard (Sigma-Aldrich) and Intact mAb Mass Check Standard (Waters)) and 1 small protein standard (β-Lactoglobulin A).     

Characterisation of intact microorganisms using electrospray ionisation mass spectrometry

We report the first application of electrospray ionisation mass spectrometry (ESI-MS) for the reproducible characterisation of strains of intact Gram-negative and Gram-positive bacteria. Electrospray ionisation was performed in both the positive and negative ion modes and the spectra obtained from Escherichia coli and Bacillus cereus were very information rich. Several of the observed negative mass ion fragments from E. coli could be assigned to specific fragmentation from bacterial phospholipids.Cluster analyses of these spectra showed that ESI-MS could be used to discriminate between these microorganisms to below species level. Therefore we conclude that ESI-MS constitutes a powerful approach to the characterisation and speciation of intact microorganisms.     

Quantifying carbohydrate-protein interactions by electrospray ionization mass spectrometry analysis

The development of analytical methods capable of characterizing carbohydrate-protein interactions, which are critical for many biological processes, represents an active area of research. Recently, the direct electrospray ionization mass spectrometry (ESI-MS) assay has emerged as a valuable tool for identifying and quantifying carbohydrate-protein complexes in vitro. The assay boasts a number of strengths, including its simplicity, speed, low level of sample consumption, and the unique ability to directly probe binding stoichiometry and to measure multiple binding equilibria simultaneously. Here, we describe the implementation of the direct ESI-MS assay for the determination of carbohydrate-protein binding stoichiometries and affinities. Common sources of error encountered with direct ESI-MS analysis of carbohydrate-protein interactions are identified along with strategies for minimizing their effects. The application of ESI-MS and a catch-and-release strategy for carbohydrate library screening are also described. The utility of the direct ESI-MS assay can be extended by combining the technique with competitive protein or ligand binding. An overview of these "indirect" ESI-MS methods is given, as well as examples of recent applications.     

Newborn mass screening and selective screening using electrospray tandem mass spectrometry in Japan

Electrospray tandem mass spectrometry was applied to detect a series of inherited metabolic disorders during a newborn-screening pilot study and a selective screening in Japan. In our mass screening of 102,200 newborns, five patients with propionic acidemia, two with methylmalonic acidemia, two with medium-chain acyl-CoA dehydrogenase deficiency, three with citrullinemia type II, and one with phenylketonuria were identified. In a selective screening of 164 patients with symptoms mainly related to hypoglycemia and/or hyperammonemia, 12 with fatty acid oxidation disorders and six with other disorders were found. The results indicated the importance of newborn screening using this technology in Japan.     

Determination of branched beta-cyclodextrin-prostaglandin complexes using electrospray ionization mass spectrometry

Mass spectral measurements by electrospray ionization mass spectrometry (ESI-MS) detected the ions of beta-cyclodextrin (betaCD) or branched betaCDs (glucosyl-, galactosyl-, mannosyl- and maltosyl-betaCD)-prostaglandins (PGs: PGA(2), PGD(2), PGE(1), PGE(2), PGF(2alpha) and PGJ(2)) complexes, i.e., betaCD-PG complexes, with a host:guest ratio of 1:1 in the negative ion mode. This is the first study to report the ions of branched betaCD-PG complexes using ESI-MS. The inclusion complexes were determined by a flow injection analysis using acetonitrile/water. We could confirm by this method the presence of a betaCD-PGE(2) complex with a host:guest ratio of 1:1 in a solution-dissolved pharmaceutical formulation consisting of betaCD-PGE(2) (Prostarmon E tablet).     

Fast proteolytic digestion coupled with organelle enrichment for proteomic analysis of rat liver

The use of an acid-labile surfactant as an alternative to urea denaturation allows for same-day proteolytic digestion and fast cleanup of cellular lysate samples. Homogenized rat liver tissue was separated into four fractions enriched in nuclei, mitochondria, microsomes (remaining organelles), and cytosol. Each subcellular fraction was then subjected to proteolytic digestion with trypsin for 2 h after denaturing with an acid-labile surfactant (ALS), separated by nanoflow reversed phase HPLC, and mass analyzed by tandem mass spectrometry in a 3-D ion trap. The results obtained from ALS denaturation for both organelle enrichment and whole cell lysate samples were comparable to those obtained from aliquots of the same samples treated by reduction, alkylation, and urea denaturation. Each method resulted in a similar number of peptides (694 for urea, 674 for ALS) and proteins (225 for urea, 229 for ALS) identified, with generally the same proteins (47% overlap) identified. As expected, organelle enrichment enabled the identification of more proteins (66% more with urea, 60% more with ALS) compared to a whole cell lysate. With organelle enrichment, the number of proteins with equal or increased sequence coverage went up by 73% with urea and 67% with ALS compared to the whole cell lysate. Additional information regarding the subcellular location of many proteins is obtained by organelle enrichment. While organelle enrichment is demonstrated with a bottom-up proteomics approach, it should be easily amenable to top-down proteomics approaches.     

An optimized protocol for metabolome analysis in yeast using direct infusion electrospray mass spectrometry

A method for the global analysis of yeast intracellular metabolites, based on electrospray mass spectrometry (ES-MS), has been developed. This has involved the optimization of methods for quenching metabolism in Saccharomyces cerevisiae and extracting the metabolites for analysis by positive-ion electrospray mass spectrometry. The influence of cultivation conditions, sampling, quenching and extraction conditions, concentration step, and storage have all been studied and adapted to allow direct infusion of samples into the mass spectrometer and the acquisition of metabolic profiles with simultaneous detection of more than 25 intracellular metabolites. The method, which can be applied to other micro-organisms and biological systems, may be used for comparative analysis and screening of metabolite profiles of yeast strains and mutants under controlled conditions in order to elucidate gene function via metabolomics. Examples of the application of this analytical strategy to specific yeast strains and single-ORF yeast deletion mutants generated through the EUROFAN programme are presented.     

General assay for sugar nucleotidyltransferases using electrospray ionization mass spectrometry

An electrospray ionization mass spectrometry-based assay has been developed to study the class of enzymes called sugar nucleotidyltransferases that couple sugar-1-phosphates and nucleotide triphosphates to form Leloir pathway glycosyl donors. The recombinant Escherichia coli and the commercially available yeast uridine-diphosphoglucose pyrophosphorylases were used as model systems. This technique allows the simultaneous and direct detection of the substrates and products without separation and, as described, is as sensitive as traditional coupled techniques. More importantly, the assay is capable of easily measuring kinetic values and inhibition constants for a range of natural and nonnatural substrates. This new assay was used to show for the first time that the reaction of the commercially available yeast uridine-diphosphoglucose pyrophosphorylase preparation is competitively inhibited by adenosine 5'-triphosphate (ATP), an observation that indicates a single active site that accepts both uridine 5'-triphosphate and ATP substrates.