Identification of protein ubiquitylation by electrospray ionization tandem mass spectrometric analysis of sulfonated tryptic peptides

We report here the application of electrospray ionization tandem mass spectrometry for the characterization of protein ubiquitylation, an important posttranslational modification of cellular proteins. Trypsin digestion of ubiquitin-conjugated proteins produces diglycine branched peptides containing the modification sites. Chemical derivatization by N-terminal sulfonation was carried out on several model peptides for the formation of a characteristic fragmentation pattern in their MS/MS analysis. The fragmentation of derivatized singly charged peptides results in a product ion distribution similar to that already observed by MALDI-TOF MS/MS. Signature fragments distinguished the diglycine branched peptides from other modified and unmodified peptides, while the sequencing product ions reveal the amino acid sequence and the location of the ubiquitylation site. Doubly charged peptide derivatives fragment in a somewhat different manner, but several fragments characteristic to diglycine branched peptides were observed under low collision energy conditions. These signature peaks can also be used to identify peptides containing ubiquitylation sites. In addition, a marker ion corresponding to a glycine-modified lysine residue produced by high-energy fragmentation provides useful information for identity verification. The method is demonstrated by the analysis of three ubiquitin-conjugated proteins using LC/MS/MS.     

Use of deuterium-labeled lysine for efficient protein identification and peptide de novo sequencing

Here, we describe a method for protein identification and de novo peptide sequencing. Through in vivo cell culturing, the deuterium-labeled lysine residue (Lys-d4) introduces a 4-Da mass tag at the carboxyl terminus of proteolytic peptides when cleaved by certain proteases. The 4-Da mass difference between the unlabeled and the deuterated lysine assigns a mass signature to all lysine-containing peptides in any pool of proteolytic peptides for protein identification directly through peptide mass mapping. Furthermore, it was used to distinguish between N- and C-terminal fragments for accurate assignments of daughter ions in tandem MS/MS spectra for sequence assignment. This technique simplifies the labeling scheme and the interpretation of the MS/MS spectra by assigning different series of fragment ions correctly and easily and is very useful in de novo peptide sequencing. We have also successfully implemented this approach to the analysis of protein mixtures derived from the human proteome.     

Single chain fragment variable recombinant antibody as a template for Fc sensors

A label free immunosensor for detection of Fc receptors expressed on cell surface was developed and characterized using a Quartz Crystal Microbalance (QCM) transducer. Taking advantage of the characteristics of single chain fragment variable (scFv) recombinant antibody and the multivalency of an antibody, the engineered recombinant scFv was immobilized onto preformed functionalized self-assembled monolayers (SAMs) template surface. The monomeric scFv can bind with the CH1 region of any rabbit IgG to form a highly oriented IgG layer with its Fc portion pointing toward a solution phase. This results in a highly oriented Fc sensor that can be used to study the thermodynamics and kinetics of binding between the Fc portion of immunoglobulin and the cell surface Fc receptor (FcR), an important area of the immune system. The Fc sensor was used to study the binding between Staphylococcus aureus and the Fc receptor on macrophage. Parallel characterization of cell surface Fc receptors in the same samples by ELISA was also performed.     

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.     

T3-sequencing: targeted characterization of the N- and C-termini of undigested proteins by mass spectrometry

A novel extension of the "top-down" approach is introduced for the selective characterization of protein termini that does not involve proteolytic digestion steps. N- and C-terminal peptides were generated from intact proteins in the mass spectrometer and further analyzed by MS/MS-an approach referred to as T(3)-sequencing. N-terminal and C-terminal fragment ion series were obtained by the pseudo-MS/MS technique in-source decay (ISD) on a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF MS). These ions provided near-terminal sequence tags from the undigested protein in the ISD spectrum acquired in reflector mode and allowed to screen for the proper processing state of the terminus with respect to a reference sequence. In the second step of T(3)-sequencing, the precursor ions, which have been generated by ISD and which included the N- or C-terminal sequence, were selected in the timed ion gate of a MALDI-TOF/TOF mass spectrometer for MS/MS analysis. These spectra allowed identification of the protein, the proper definition of both termini, and allowed confirmation of suspected terminal modifications. T(3)-Sequencing appears to be an alternative to classical Edman sequencing, which is fast and even permits the analysis of N-terminally blocked proteins and their C-terminus.     

Proteome-wide identification of proteins and their modifications with decreased ambiguities and improved false discovery rates using unique sequence tags

Identifying proteins and their modification states and with known levels of confidence remains as a significant challenge for proteomics. Random or decoy peptide databases are increasingly being used to estimate the false discovery rate (FDR), e.g., from liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses of tryptic digests. We show that this approach can significantly underestimate the FDR and describe an approach for more confident protein identifications that uses unique partial sequences derived from a combination of database searching and amino acid residue sequencing using high-accuracy MS/MS data. Applied to a Saccharomyces cerevisiae tryptic digest, the approach provided 3 132 confident peptide identifications ( approximately 5% modified in some fashion), covering 575 proteins with an estimated zero FDR. The conventional approach provided 3 359 peptide identifications and 656 proteins with 0.3% FDR based upon a decoy database analysis. However, the present approach revealed approximately 5% of the 3 359 identifications to be incorrect and many more as potentially ambiguous (e.g., due to not considering certain amino acid substitutions and modifications). In addition, 677 peptides and 39 proteins were identified that had been missed by conventional analysis, including nontryptic peptides, peptides with a variety of expected/unexpected chemical modifications, known/unknown post-translational modifications, single nucleotide polymorphisms or gene encoding errors, and multiple modifications of individual peptides.     

Divinyl sulfone as a postdigestion modifier for enhancing the a(1) Ion in MS/MS and postsource decay: potential applications in proteomics

Divinyl sulfone reacts at pH 8-9 with the alpha-amino groups of N-terminal residues, proline, the epsilon-amino groups of lysine, and the histidine side chains of peptides. This reaction leads to great enhancement of the abundance of the normally weak or missing "a(1)" fragment ion in MS/MS analysis defining the N-terminal residue of a peptide in a digest. This provides "one-step Edman-like" information that, together with a fairly accurately determined mass, often enables one to correctly identify a protein or family of proteins. The applicability of this procedure in proteomics was demonstrated with several peptides and tryptic digests of protein mixtures by LC-MS/MS experiments using a QTOF and MALDI-PSD analyses. Advantages of this approach are its simple chemistry, retention of charge multiplicity, and possibly, shortening of database search time. Used with other MS/MS data, it provides higher confidence in the scores and identification of a protein found in peptide mass fingerprinting. Moreover, this approach has an advantage in "de novo" sequencing due to its ability to decipher the first amino acid of a peptide whose information is normally unavailable in MS/MS spectra.     

In vivo deamidation characterization of monoclonal antibody by LC/MS/MS

The spontaneous nonenzymatic deamidation of glutaminyl and asparaginyl residues of peptides and proteins has been observed both in vitro and in vivo. Deamidation may change the structure and function of a peptide or protein, potentially resulting in decreased bioactivity, as well as alterations in pharmacokinetics and antigenicity of the protein pharmaceutical. Therefore, it is necessary to monitor the effect of storage and formulation conditions on deamidation of a protein drug candidate. Of particular interest is the investigation of in vivo deamidation mechanisms of protein drug candidates. Several methods are available to characterize the deamidation of peptides and proteins. We present here a LC/MS/MS method used to evaluate the deamidation of an antibody after in vivo administration. A humanized monoclonal IgG1 antibody (MAb) has several "hot spots" for spontaneous deamidation. One site, amino acid residue Asn55 located in the CDR2 region of the heavy chain, is of particular interest since deamidation at this site greatly decreases the binding activity. MAb was administered to cynomolgus monkeys by intravenous and subcutaneous routes. At various times after dosing, monkey serum was prepared and MAb captured by the immobilized antigen or a goat anti-human IgG Fcgamma antibody. The captured MAb was treated with trypsin followed by endoproteinase Glu-C. The digests were separated on RP-HPLC and analyzed by MS/MS on Q-Tof Global mass spectrometer. Using this method, we were able to determine the deamidation half-life of amino acid residue Asn55 in vivo and the ratio of the deamidated derivatives, i.e., isoAsp55 and Asp55. The method is rapid and sensitive with low-nanogram quantities of protein detected in the biological matrix.     

Utility of immonium ions for assignment of epsilon-N-acetyllysine-containing peptides by tandem mass spectrometry

Tandem mass spectrometry (MS/MS) is a powerful tool for characterization of post-translationally modified proteins, including epsilon-N-acetyllysine-containing species. Previous reports indicate that epsilon-N-acetyllysine immonium ions are useful marker ions for peptides containing epsilon-N-acetyllysine, but the specificity and sensitivity of these ions for assignment of lysine acetylation by MS/MS have not been studied in detail. We investigated MS/MS data sets of 172 epsilon-N-acetyllysine tryptic peptides and 268 nonacetylated tryptic peptides to establish the utility and reliability of epsilon-N-acetyllysine immonium ions for identification and validation of acetylated peptides. Our analysis shows that the immonium ion at m/z 143 lacks specificity for lysine-acetylated peptides, whereas the derivative at m/z 126 is highly specific (98.1%). We also studied the positional effect of the epsilon-N-acetyllysine on the intensity of observed acetyllysine immonium ions. We observed an increase in acetyllysine immonium ion intensities when the acetylated lysine was N-terminally positioned in the peptide as compared to internal positions. Based on these observations we propose a validation scheme for unambiguous assignment of acetyllysine-containing peptides by MS/MS. Our analysis of epsilon-N-acetyllysine immonium ions provide a framework for investigation of MS/MS marker ion specificity and sensitivity that can be applied in studies of other types of post-translational modifications.     

Hunting for "key residues" in the modeling of globular protein folding: an artificial neural network-based approach

An approach to modeling globular protein folding based on artificial neural networks (ANNs) is presented. This approach, that can be regarded as an inverse protein folding problem, investigates whether and when a protein fragment needs a specific residue in the center of its primary structure as a necessary condition to fold as observed. To perform this analysis, an ANN has been trained on a set of 55 proteins, searching for a relation between protein fragments modeled by 13/spl alpha/ torsion angles and the residue corresponding to the central /spl alpha/ torsion angle of the fragment. The results obtained show that only Asp, Gly, Pro, Ser and Val residues are often a necessary, even though not sufficient, condition to obtain a specific folded fragment structure, playing therefore, the role of "key residue" of this fragment.     

"Proteotyping": population proteomics of human leukocytes using top down mass spectrometry

Characterizing combinations of coding polymorphisms (cSNPs), alternative splicing and post-translational modifications (PTMs) on a single protein by standard peptide-based proteomics is challenging owing to <100% sequence coverage and the uncoupling effect of proteolysis on such variations >10-20 residues apart. Because top down MS measures the whole protein, combinations of all the variations affecting primary sequence can be detected as they occur in combination. The protein form generated by all types of variation is here termed the "proteotype", akin to a haplotype at the DNA level. Analysis of proteins from human primary leukocytes harvested from leukoreduction filters using a dual on-line/off-line top down MS strategy produced >600 unique intact masses, 133 of which were identified from 67 unique genes. Utilizing a two-dimensional platform, termed multidimensional protein characterization by automated top down (MudCAT), 108 of the above protein forms were subsequently identified in the absence of MS/MS in 4 days. Additionally, MudCAT enables the quantitation of allele ratios for heterozygotes and PTM occupancies for phosphorylated species. The diversity of the human proteome is embodied in the fact that 32 of the identified proteins harbored cSNPs, PTMs, or were detected as proteolysis products. Among the information were three partially phosphorylated proteins and three proteins heterozygous at known cSNP loci, with evidence for non-1:1 expression ratios obtained for different alleles.     

Characterization of polyubiquitin chain structure by middle-down mass spectrometry

Ubiquitin (Ub) is a 76 amino acid polypeptide that modifies a wide range of proteins in the types of monomer or polymers, and functional consequence of ubiquitination is modulated by the length and topologies of polyUb chains. Whereas polyUb chains are usually analyzed by fully trypsin digestion and mass spectrometry (MS), we present here a middle-down strategy to characterize the structure of polyUb chains by high-resolution mass spectrometry (MS). Under optimized condition, native folded polyUb is partially trypsinized exclusively at the R74 residue, generating a large Ub fragment (1-74 residues termed UbR74) and its ubiquitinated form with a diglycine tag (UbR74-GG). The molar ratio between UbR74 and UbR74-GG reflects the length of homogeneous polyUb chains (i.e., 1:1 for the dimer, 1:2 for the trimer, 1:3 for the tetramer, and so on). Moreover, lysine residues in ubiquitin used for chain linkages are detectable by MS/MS and MS/MS/MS of large GG-tagged Ub fragments. The strategy was validated using a number of ubiquitin polymers, including K48-linked human di-Ub, K63-linked human tetra-Ub, as well as His-tagged polyUb chains purified from yeast under native condition. The potential of this strategy to analyze polyUb chains with mixed linkages (e.g., forked chains) is also discussed. Together, this middle-down MS strategy provides a novel complementary method for studying the length and linkages of complex polyUb chain structures.     

Monoclonal antibody cocktail as an enrichment tool for acetylome analysis

The availability and robustness of methods to analyze phosphorylated proteins has greatly expanded our knowledge of phosphorylation based cell signaling. A key ingredient to the success of these studies is the ability to enrich phosphopeptides using antibodies or other chemical approaches. Most other post-translational modifications, such as lysine acetylation, are still poorly characterized because of the lack of availability of such enrichment methods. Recently, some groups have reported identification of acetylation sites in a global fashion by enriching acetylated peptides with a polyclonal antibody from a single source that was raised against pan-acetylated lysine. Instead of the use of this polyclonal antibody, we used a cocktail of monoclonal antibodies where each was directed against acetylated lysine in different contexts. Using high resolution Fourier transform mass spectrometry, we observed that the majority of acetylated lysine residues identified using the monoclonal antibody cocktail were distinct from those enriched by the polyclonal antibody used by the other groups. Our study demonstrates that immunoaffinity enrichment of acetylated peptides is somewhat limited by substrate specificity and that an optimal yield of enrichment can be achieved by employing a broader array of affinity reagents.     

Identification of intact proteins in mixtures by alternated capillary liquid chromatography electrospray ionization and LC ESI infrared multiphoton dissociation Fourier transform ion cyclotron resonance mass spectrometry.

Here we propose a novel method for rapidly identifying proteins in complex mixtures. A list of candidate proteins (including provision for posttranslational modifications) is obtained by database searching, within a specified mass range about the accurately measured mass (e.g., +/- 0.1 Da at 10 kDa) of the intact protein, by capillary liquid chromatography electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (LC ESI FT-ICR MS). On alternate scans, LC ESI infrared multiphoton dissociation (IRMPD) FT-ICR MS yields mostly b and y fragment ions for each protein, from which the correct candidate is identified as the one with the highest "hit" score (i.e., most b and y fragments matching the candidate database protein amino acid sequence masses) and sequence "tag" score (based on a series of fragment sequences differing in mass by 1 or 2 amino acids). The method succeeds in uniquely identifying each of a mixture of five proteins treated as unknowns (melittin, ubiquitin, GroES, myoglobin, carbonic anhydrase II), from more than 1000 possible database candidates within a +/- 500 Da mass window. We are also able to identify posttranslational modifications of two of the proteins (mellitin and GroES). The method is simple, rapid, and definitive and is extendable to a mixture of affinity-selected proteins, to identify proteins with a common biological function.     

Noncompetitive detection of low molecular weight peptides by open sandwich immunoassay

Small peptides with less than 1000 in molecular weight are not considered amenable to sandwich immunoassays due to their difficulty of simultaneous recognition by two antibodies. As an alternative, we attempted noncompetitive detection of small peptides by open sandwich enzyme-linked immunosorbent assay (OS-ELISA) utilizing the antigen-induced enhancement of antibody VH/VL interaction. Taking fragments of human osteocalcin (BGP), a major non-collagen peptide produced in bone, as model peptides, OS immunoassay was performed using the cloned VH and VL cDNAs from two anti-BGP monoclonal antibodies either recognizing the N- or C-terminal fragment, respectively. When the clones were used for OS-ELISA with immobilized VL fragment and phage-displayed VH fragment, enhanced VH/VL interaction upon BGP addition was observed. Especially the clone for the C-terminal fragment showed a superior detection limit as well as a wider working range than those of competitive assay. The result was reproduced with purified VH-alkaline phosphatase and MBP-VL fusion proteins, where the latter was directly immobilized onto the microplate wells. The minimum detectable fragment was the hexamer including the C-terminus. This simple approach with a single monoclonal antibody with a short measurement time may prove a useful tool in immunodiagnostics as well as in proteomics research.     

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.     

Characterization of transthyretin variants in familial transthyretin amyloidosis by mass spectrometric peptide mapping and DNA sequence analysis

Transthyretin (TTR) is a 127-amino acid residue transport protein. In plasma, TTR exists as a tetramer and binds the hormone thyroxine and the retinol-binding protein-vitamin A complex. Amino acid substitutions in TTR are hypothesized to destabilize the tetramer and cause the protein to form intermediates that self-associate into amyloid fibrils. Familial transthyretin amyloidosis (ATTR) is associated with extracellular deposition of wild-type TTR, its variants or fragments as amyloid fibrils in various tissues and organs. A definitive diagnosis of ATTR depends on the detection and identification of TTR variants. Electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS), in combination with trypsin digestion, have been shown to be powerful tools in characterizing TTR variants. Typically, TTR or its tryptic digest is analyzed by MALDI-TOF MS, liquid chromatography ESI MS, or both. Analysis of tryptic digests by MALDI-TOF MS does not provide enough sequence coverage in TTR to identify all possible modifications. To improve sequence coverage, aliquots of immunoprecipitated TTR samples were digested with trypsin, lysyl endopeptidase Lys-C, or endoproteinase Asp-N. Identification of the peptides from each digest by MALDI-TOF MS provided preliminary information about the sites and mass shifts due to amino acid substitutions from genetic mutations and to posttranslational modifications. The location and identity of the modifications in the variant proteins were then confirmed by tandem mass spectrometry, accurate mass measurements, and direct DNA sequence analysis. Using these methodologies, we achieved 100% sequence coverage. The detection of two nonpathologic variants (Thr119Met and Gly6Ser) and four pathologic variants (Phe64Leu, Asp38Ala, Phe44Ser, and previously unreported Trp41Leu) are described as illustrations of this approach.     

Fully automated chemiluminescence immunoassay of insulin using antibody-protein A-bacterial magnetic particle complexes

We report a fully automated sandwich immunoassay for the determination of human insulin using antibody-protein A-bacterial magnetic particle (BMP) complexes and an alkaline phosphatase-conjugated secondary antibody. BMPs bearing protein A-MagA inserted on the external surface of the membrane were prepared in the Magnetospirillum sp. AMB-1 transconjugant for a protein A-magA fusion gene. MagA protein was used as an anchor to attach protein A onto the membrane. Protein A-BMP complexes harvested from transconjugant AMB-1 were subsequently complexed with anti-human insulin antibodies by specific binding between the Z domain of protein A and the Fc component of IgG to form the antibody-protein A-BMP complexes. The complexes were quite monodisperse after the binding of the antibody. The BMPs' monodispersity resulted in high signal and low noise in the immunoassay. The luminescence intensity ((kilocounts/s)/microg of antibody) from antibody-protein A-BMP complexes after immunoreaction was higher than that from BMPs chemically conjugated to an antibody. This was explained by a difference in dispersion. The fully automated sandwich immunoassay system using antibody-protein A-BMP complexes made possible precise assays of human insulin in serum.     

Top-down and bottom-up mass spectrometric characterization of human myoglobin-centered free radicals induced by oxidative damage

In an effort to determine the utility of top-down mass spectrometric methodologies for the characterization of protein radical adducts, top-down approaches were investigated and compared to the traditional bottom-up approaches. Specifically, the nature of the radicals on human myoglobin induced by the addition of hydrogen peroxide and captured by the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was investigated. The most abundant ion observed in the electrospray mass spectrum of this reaction mixture corresponds in mass to the human myoglobin plus one DMPO molecule. In addition, a second ion of lower abundance is observed, which corresponds to a second DMPO molecule being trapped on myoglobin. Top-down analyses using Fourier transform ion cyclotron resonance mass spectrometry can be used to characterize proteins and, thus, were performed on several different charge-state ions of both the native and the mono-DMPO nitrone adduct of human myoglobin. Data produced from the top-down analyses are very complex yet information rich. In the case of DMPO-modified human myoglobin, the top-down data localized the DMPO spin trap to residues 97-110 of the myoglobin. The observation of the y43+5 fragment ion arising from C-terminal cleavage to the cysteine-110 residue in the MS/MS spectrum of DMPO-modified myoglobin and not in the unmodified myoglobin implicates a change to this residue, specifically, DMPO adduction. On the other hand, using the traditional bottom-up approach of peptide mapping and MS sequencing methodologies, two DMPO radical adducts on human myoglobin were identified, Cys-110 and Tyr-103. The bottom-up approach is more proven and robust than the top-down methodologies. Nonetheless, the bottom-up and top-down approaches to protein characterization are complementary rather than competitive approaches with each having its own utility.     

Improved mass spectrometric characterization of protein glycosylation reveals unusual glycosylation of maize-derived bovine trypsin

Although bottom-up proteomics using tryptic digests is widely used to locate post-translational modifications (PTM) in proteins, there are cases where the protein has several potential modification sites within a tryptic fragment and MS(2) strategies fail to pinpoint the location. We report here a method using two proteolytic enzymes, trypsin and pepsin, in combination followed by tandem mass spectrometric analysis to provide fragments that allow one to locate the modification sites. We used this strategy to find a glycosylation site on bovine trypsin expressed in maize (TrypZean). Several glycans are present, and all are attached to a nonconsensus N-glycosylation site on the protein.