基于ProteinGoggle 2.0的组蛋白H4蛋白质变体的自上而下表征

由于大量可能蛋白质变体以及每一个翻译后修饰大量可能位点的存在,核心组蛋白上密集的组合式翻译后修饰的自上而下表征一直是一个巨大的分析挑战。结合高分辨串级质谱,基于同位素质荷比和轮廓指纹比对的整体蛋白质数据库搜索引擎ProteinGoggle 2.0在组蛋白翻译后修饰的自上而下鉴定方面拥有诸多独特的优势。该文报道ProteinGoggle 2.0对HeLa核心组蛋白H4的数据库搜索及蛋白质变体的鉴定结果。基于从UniProt网站下载的人类核心组蛋白H4的纯文本文件和“鸟枪法”注释,ProteinGoggle 2.0首先创建包含所有可能蛋白质变体的理论数据库;从纯文本文件中提取的信息主要是氨基酸序列、可能的翻译后修饰（单甲基化、二甲基化、三甲基化、乙酰化和磷酸化）及氨基酸变异（A77→P）。在控制质谱水平假阳性率低于1%的前提下,共鉴定到426个蛋白质变体,这是目前为止H4蛋白质变体的最全报道。这些ProteinGoggle 2.0鉴定到的H4蛋白质变体也与之前报道的ProSightPC 2.0的鉴定结果进行了肩并肩比较。总而言之,ProteinGoggle 2.0可以对具有复杂组合修饰及氨基酸变异的蛋白质组进行数据库搜索和蛋白质变体鉴定。     

Peptide and protein characterization by high-rate electron capture dissociation Fourier transform ion cyclotron resonance mass spectrometry

The analytical utility of the electron capture dissociation (ECD) technique, developed by McLafferty and co-workers, has substantially improved peptide and protein characterization using Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). The limitations of the first ECD implementations on commercial instruments were eliminated by the employment of low-energy electron-injection systems based on indirectly heated dispenser cathodes. In particular, the ECD rate and reliability were greatly increased, enabling the combination of ECD/FTICR-MS with on-line liquid separation techniques. Further technique development allowed the combination of two rapid fragmentation techniques, high-rate ECD and infrared multiphoton dissociation (IRMPD), in a single experimental configuration. Simultaneous and consecutive irradiations of trapped ions with electrons and photons extended the possibilities for ion activation/dissociation and led to improved peptide and protein characterization. The application of high-rate ECD/FTICR-MS has demonstrated its power and unique capabilities in top-down sequencing of peptides and proteins, including characterization of post-translational modifications, improved sequencing of peptides with multiple disulfide bridges and secondary fragmentation (w-ion formation). Analysis of peptide mixtures has been accomplished using high-rate ECD in bottom-up mass spectrometry based on mixture separation by liquid chromatography and capillary electrophoresis. This paper summarizes the current impact of high-rate ECD/FTICR-MS for top-down and bottom-up mass spectrometry of peptides and proteins.     

Emerging methods in proteomics: top-down protein characterization by multistage tandem mass spectrometry

"Top-down" mass spectrometry methods have emerged as an attractive alternative to conventional "bottom-up" approaches for the comprehensive characterization of co- and post-translational protein modifications. Here we present a brief overview of current strategies employed for top-down protein characterization and discuss the key technical challenges and solutions associated with their implementation on a range of mass spectrometry instrument platforms. For more specific details regarding the individual strategies described herein, interested readers are referred to the references cited at the end of this article.     

Precursor ion independent algorithm for top-down shotgun proteomics

We present a precursor ion independent top-down algorithm (PIITA) for use in automated assignment of protein identifications from tandem mass spectra of whole proteins. To acquire the data, we utilize data-dependent acquisition to select protein precursor ions eluting from a C4-based HPLC column for collision induced dissociation in the linear ion trap of an LTQ-Orbitrap mass spectrometer. Gas-phase fractionation is used to increase the number of acquired tandem mass spectra, all of which are recorded in the Orbitrap mass analyzer. To identify proteins, the PIITA algorithm compares deconvoluted, deisotoped, observed tandem mass spectra to all possible theoretical tandem mass spectra for each protein in a genomic sequence database without regard for measured parent ion mass. Only after a protein is identified, is any difference in measured and theoretical precursor mass used to identify and locate post-translation modifications. We demonstrate the application of PIITA to data generated via our wet-lab approach on a Salmonella typhimurium outer membrane extract and compare these results to bottom-up analysis. From these data, we identify 154 proteins by top-down analysis, 73 of which were not identified in a parallel bottom-up analysis. We also identify 201 unique isoforms of these 154 proteins at a false discovery rate (FDR) of <1%.     

Mass spectrometry in biodefense

Potential agents for biological attacks include both microorganisms and toxins. In mass spectrometry (MS), rapid identification of potential bioagents is achieved by detecting the masses of unique biomarkers, correlated to each agent. Currently, proteins are the most reliable biomarkers for detection and characterization of both microorganisms and toxins, and MS-based proteomics is particularly well suited for biodefense applications. Confident identification of an organism can be achieved by top-down proteomics following identification of individual protein biomarkers from their tandem mass spectra. In bottom-up proteomics, rapid digestion of intact protein biomarkers is again followed by MS/MS to provide unambiguous bioagent identification and characterization. Bioinformatics obviates the need for culturing and rigorous control of experimental variables to create and use MS fingerprint libraries for various classes of bioweapons. For specific applications, MS methods, instruments and algorithms have also been developed for identification based on biomarkers other than proteins and peptides.     

An algorithm for identifying multiply modified endogenous proteins using both full-scan and high-resolution tandem mass spectrometric data

Mass spectrometry based proteomic experiments have advanced considerably over the past decade with high-resolution and mass accuracy tandem mass spectrometry (MS/MS) capabilities now allowing routine interrogation of large peptides and proteins. Often a major bottleneck to 'top-down' proteomics, however, is the ability to identify and characterize the complex peptides or proteins based on the acquired high-resolution MS/MS spectra. For biological samples containing proteins with multiple unpredicted processing events, unsupervised identifications can be particularly challenging. Described here is a newly created search algorithm (MAR) designed for the identification of experimentally detected peptides or proteins. This algorithm relies only on predefined list of 'differential' modifications (e.g. phosphorylation) and a FASTA-formatted protein database, and is not constrained to full-length proteins for identification. The algorithm is further powered by the ability to leverage identified mass differences between chromatographically separated ions within full-scan MS spectra to automatically generate a list of likely 'differential' modifications to be searched. The utility of the algorithm is demonstrated with the identification of 54 unique polypeptides from human apolipoprotein enriched from the high-density lipoprotein particle (HDL), and searching time benchmarks demonstrate scalability (12 high-resolution MS/MS scans searched per minute with modifications considered). This parallelizable algorithm provides an additional solution for converting high-quality MS/MS data of multiply processed proteins into reliable identifications.     

A robust two-dimensional separation for top-down tandem mass spectrometry of the low-mass proteome

For fractionation of intact proteins by molecular weight (MW), a sharply improved two-dimensional (2D) separation is presented to drive reproducible and robust fractionation before top-down mass spectrometry of complex mixtures. The “GELFrEE” (i.e., gel-eluted liquid fraction entrapment electrophoresis) approach is implemented by use of Tris-glycine and Tris-tricine gel systems applied to human cytosolic and nuclear extracts from HeLa S3 cells, to achieve a MW-based fractionation of proteins from 5 to >100 kDa in 1 h. For top-down tandem mass spectroscopy (MS/MS) of the low-mass proteome (5–25 kDa), between 5 and 8 gel-elution (GE) fractions are sampled by nanocapillary-LC-MS/MS with 12 or 14.5 tesla Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers. Single injections give about 40 detectable proteins, about half of which yield automated ProSight identifications. Reproducibility metrics of the system are presented, along with comparative analysis of protein targets in mitotic versus asynchronous cells. We forward this basic 2D approach to facilitate wider implementation of top-down mass spectrometry and a variety of other protein separation and/or characterization approaches.     

Fast Characterization of Fc-Containing Proteins by Middle-Down Mass Spectrometry Following IdeS Digestion

The rapid growth of biotherapeutics (monoclonal antibodies and antibody derivatives) demands improved techniques for their quality control and batch-to-batch study. Traditionally, the top-down and bottom-up mass spectrometric approaches may be good options, but they have their share of drawbacks. The middle-down technology has unique superiorities for fast characterization of very large proteins. In this article, we report a systematic approach to characterize Fc-containing proteins (antibody, Fc fusion protein, and antibody–drug conjugate) by middle-down mass spectrometry following IdeS proteolytic digestion. We characterized the main protein modifications including glycosylation, glycation, and other modifications with a relatively small proportion, and results were consistent with free glycan profiling analysis. Meanwhile, the O-acetylated sialic acid modification of cetuximab was reported for the first time; its potential roles in biotherapeutics need further study. The middle-down technology following IdeS proteolytic digestion could be used not only for analyzing the modifications of Fc-containing proteins at the subunit level but also for characterizing the potential modifications at early development stages.

     

A top-down LC-FTICR MS-based strategy for characterizing oxidized calmodulin in activated macrophages

A liquid chromatography-mass spectrometry (LC-MS)-based approach for characterizing the degree of nitration and oxidation of intact calmodulin (CaM) has been used to resolve ～250 CaM oxiforms using only 500 ng of protein. The analysis was based on high-resolution data of the intact CaM isoforms obtained by Fourier-transform ion cyclotron resonance mass spectrometry (FTICR MS) coupled with an on-line reversed-phase LC separation. Tentative identifications of post-translational modifications (PTMs), such as oxidation or nitration, have been assigned by matching observed protein mass to a database containing all theoretically predicted oxidation products of CaM and verified through a combination of tryptic peptide information (generated from bottom-up analyses) and on-line collisionally induced dissociation (CID) tandem mass spectrometry (MS/MS) at the intact protein level. The reduction in abundance and diversity of oxidatively modified CaM (i.e., nitrated tyrosines and oxidized methionines) induced by macrophage activation has been explored and semiquantified for different oxidation degrees (i.e., no oxidation, moderate, and high oxidation). This work demonstrates the power of the top-down approach to identify and quantify hundreds of combinations of PTMs for single protein target such as CaM and implicate competing repair and peptidase activities to modulate cellular metabolism in response to oxidative stress.     

Confident assignment of intact mass tags to human salivary cystatins using top-down Fourier-transform ion cyclotron resonance mass spectrometry

A hybrid linear ion-trap Fourier-transform ion cyclotron resonance mass spectrometer was used for top-down characterization of the abundant human salivary cystatins, including S, S1, S2, SA, SN, C, and D, using collisionally activated dissociation (CAD) after chromatographic purification of the native, disulfide intact proteins. Post-translational modifications and protein sequence polymorphisms arising from single nucleotide polymorphisms (SNPs) were assigned from precursor and product ion masses at a tolerance of 10 ppm, allowing confident identification of individual intact mass tags. Cystatins S, S1, S2, SA, and SN were cleaved of a N-terminal 20 amino acid signal peptide and cystatin C a 26-residue peptide, to yield a generally conserved N-terminus. In contrast, cystatin D isoforms with 24 and 28 amino acid residue N-terminal truncations were found such that their N-termini were not conserved. Cystatin S1 was phosphorylated at Ser3, while S2 was phosphorylated at Ser1 and Ser3, in agreement with previous work. Both cystatin D isoforms carried the polymorphism C46R (SNP: rs1799841). The 14,328 Da isoform of cystatin SN previously assigned with polymorphism P31L due to a SNP (rs2070856) was found only in whole saliva. Parotid secretions contained no detectable cystatins while whole saliva largely mirrored the contents of submandibular/sublingual (SMSL) secretions. With fully characterized cystatin intact mass tags it will now be possible to examine the correlation between the abundance of these molecules and human health and disease.     

Preconcentration and detection of the phosphorylated forms of cardiac troponin I in a cascade microchip by cationic isotachophoresis

This paper describes the detection of a cardiac biomarker, cardiac troponin I (cTnI), spiked into depleted human serum using cationic isotachophoresis (ITP) in a 3.9 cm long poly(methyl methacrylate) (PMMA) microfluidic channel. The microfluidic chip incorporates a 100× cross-sectional area reduction, including a 10× depth reduction and a 10× width reduction, to increase sensitivity during ITP. The cross-sectional area reductions in combination with ITP allowed visualization of lower concentrations of fluorescently labeled cTnI. ITP was performed in both "peak mode" and "plateau mode" and the final concentrations obtained were linear with initial cTnI concentration. We were able to detect and quantify cTnI at initial concentrations as low as 46 ng mL(-1) in the presence of human serum proteins and obtain cTnI concentrations factors as high as ~ 9000. In addition, preliminary ITP experiments including both labeled cTnI and labeled protein kinase A (PKA) phosphorylated cTnI were performed to visualize ITP migration of different phosphorylated forms of cTnI. The different phosphorylated states of cTnI formed distinct ITP zones between the leading and terminating electrolytes. To our knowledge, this is the first attempt at using ITP in a cascade microchip to quantify cTnI in human serum and detect different phosphorylated forms.     

Quantitative in vitro kinase reaction as a guide for phosphoprotein analysis by mass spectrometry

A simple calculation using the radioactive decay of (32)P incorporated into a protein during in vitro kinase reactions is described that allows the overall stoichiometry of phosphorylation for the substrate protein or peptide to be calculated. Prior to using techniques such as diagnostic ion scanning to identify the molecular weight of an unknown phosphopeptide in a complex mixture followed by tandem mass spectrometry (MS/MS) to locate the phosphorylated residue within the phosphopeptide, such calculations are predictive of the chances for successful characterization by these methods. An example of estimating the stoichiometry of peptide phosphorylation will be presented along with calculations that predict when adequate phosphopeptide is present in any given spot on the thin-layer chromatography (TLC) plates used for two-dimensional phosphopeptide (2DPP) mapping to allow extraction and complete characterization by MS/MS.     

Identification of protein phosphorylation sites within Ser/Thr-rich cluster domains using site-directed mutagenesis and hybrid linear quadrupole ion trap Fourier transform ion cyclotron resonance mass spectrometry

We describe a method for the analysis of multi-site phosphorylation in serine/threonine (Ser/Thr)-rich protein sequences. Site-specific mutagenesis was used to introduce tryptic cleavage sites in the serine glutamine/threonine glutamine cluster domain (SCD) of the human checkpoint protein kinase (Chk2). The mutant proteins were shown to autophosphorylate on residues that are inducibly phosphorylated when mammalian cells are exposed to ionizing radiation (serine 33/35, serine 516, threonine 68 and threonine 432). Five Ser/Thr clusters within the SCD were flanked by arginine or lysine residues to produce tryptic peptides for nanospray liquid chromatography (nanoLC)/linear quadrupole ion trap Fourier transform ion cyclotron resonance mass spectrometry. Phosphorylation sites were assigned using accurate-mass-driven analysis and interpretation of low-energy collision-induced dissociation spectra acquired in the ion trap. In addition to verifying known phosphorylation sites, seventeen novel sites were identified within the SCD of Chk2. The approach should be applicable to other O-linked post-translational modifications that occur in proteins with Ser/Thr-rich sequences.     

Top-down mass spectrometry: recent developments, applications and perspectives

Top-down mass spectrometry is an emerging approach for the analysis of intact proteins. The term was coined as a contrast with the better-established, bottom-up strategy for analysis of peptide fragments derived from digestion, either enzymatically or chemically, of intact proteins. Although the term top-down originates from proteomics, it can also be applied to mass spectrometric analysis of intact large biomolecules that are constituents of protein assemblies or complexes. Traditionally, mass spectrometry has usually started with intact molecules, and in this regard, top-down approaches reflect the spirit of mass spectrometry. This article provides an overview of the methodologies in top-down mass spectrometry and then reviews applications covering protein posttranslational modifications, protein biophysics, DNAs/RNAs, and protein assemblies. Finally, challenges and future directions are discussed.     

Mass spectral determination of skeletal/cardiac actin isoform ratios in cardiac muscle

Skeletal and cardiac muscle contains actin isoforms that vary by two juxtaposed amino acids and two amino acid substitutions (Met299Leu and Ser358Thr). This close sequence homology does not allow cardiac and skeletal actin isoforms to be resolved in traditional SDS-PAGE analysis as the molecular weights (Deltamass = 32 Da) are not significantly different and the pIs are identical (5.2). Although cardiac actin is the predominant form in cardiac muscle, there appears to be a specific skeletal/cardiac actin ratio in a normal heart that may vary in a compromised or diseased heart. In an effort to ascertain the validity of this hypothesis we developed a mass spectrometric technique to measure the ratio of skeletal to cardiac actin. The technique involves purification of muscle actin and subsequent liquid chromatography coupled with electrospray ionization Fourier transform ion cylcotron resonance (LC/FTICR-MS) mass spectrometry. A 7 Tesla FTICR mass spectrometer was utilized to compare skeletal/cardiac actin isoform ratios. Additionally, a new dual electrospray ionization source was employed to determine accurate masses of the alpha-skeletal and alpha-cardiac actins.     

Precise proteomic identification using mass spectrometry coupled with stable isotope labeling

Stable isotope labeling (SIL) can assist mass spectrometry to improve its specificity and throughput in protein identification, de novo sequencing, and characterization of post translational modifications. This Education article summarizes the unique characteristics of stable isotope-assisted mass spectrometry for accurate protein identification without requirements for ultrahigh mass accuracy. Some applications are discussed here to demonstrate the general experimental procedures, data interpretation, and the improvements in accuracy and throughput compared to the use of mass spectrometry alone.     

Detailed structural elucidation of polyesters and acrylates using Fourier transform mass spectrometry

The detailed structural characterization of complex polymer architectures, like copolymers and polymer mixtures, by mass spectrometry presents a challenge. Even though soft ionization analyses revolutionized the characterization of large molecules and provided a means for determining the polymer’s molecular weight distribution, polydispersity, and end groups, full microstructure elucidation and monomer sequencing by soft ionization alone is not possible. The combination of high-resolution Fourier transform mass spectrometry (FTMS) and tandem mass spectrometry (MSⁿ) provides a powerful analytical tool for addressing these challenges. This tool was used in our work to separate and identify the products of polymerization between 12-hydroxystearic acid (HSA) and stearic acid (SA), to provide precise information about the exact location of caprolactones on the Tris(2-hydroxyethyl)isocyanurate (THEIC) molecule, and to sequence a glycidyl methacrylate/methyl methacrylate (GMA/MMA) copolymer. The results highlight the value of ultrahigh resolution and tandem mass spectrometry for fine structural characterization and sequencing of polymers.     

Archived polyacrylamide gels as a resource for proteome characterization by mass spectrometry

Mass spectrometry was applied to identify protein spots excised from an archived two-dimensional polyacrylamide gel that had been dried and stored for eight years at room temperature. All proteins were successfully identified. Detailed characterization of protein digests by matrix-assisted laser desorption/ionization (MALDI) peptide mapping, nanoelectrospray tandem mass spectrometry and MALDI-quadrupole time-of-flight mass spectrometry revealed no evidence of protein degradation or modifications that could hamper identification of proteins in a sequence database. The experiment with a model protein demonstrated that the pattern of tryptic peptides and the yield of individual peptides were not noticeably changed in the in-gel digest of the archived protein spot compared to the digest of the spot excised from a fresh gel. Thus, the characterization of "archived proteomes" has the potential to advance proteomic research without repeating "wet" biochemistry experiments, that had been perfected in the laboratory years ago.     

Electron detachment dissociation of dermatan sulfate oligosaccharides

The structural characterization of glycosaminoglycans (GAG) oligosaccharides has been a long-standing challenge in the field of mass spectrometry. In this work, we present the application of electron detachment dissociation (EDD) Fourier transform mass spectrometry to the analysis of dermatan sulfate (DS) oligosaccharides up to 10 residues long. The EDD mass spectra of DS oligosaccharides were compared with their infrared multiphoton dissociation (IRMPD) mass spectra. EDD produces more abundant fragmentation than IRMPD with far less loss of SO3 from labile sulfate modifications. EDD cleaves all glycosidic bonds, yielding both conventional glycosidic bond fragmentation as well as satellite peaks resulting from the additional loss of 1 or 2 hydrogen atoms. EDD also yields more cross-ring fragmentation than IRMPD. For EDD, abundant cross-ring fragmentation in the form of A- and X-ions is observed, with 1,5Xn cleavages occurring for all IdoA residues and many of the GalNAc4S residues, except at the reducing and nonreducing ends. In contrast, IRMPD produces only A-type cross-ring fragmentation for long oligosaccharides (dp6-dp10). As all the structurally informative fragment ions observed by IRMPD appear as a subset of the peaks found in the EDD mass spectrum, EDD shows great potential for the characterization of GAG oligosaccharides using a single tandem mass spectrometry experiment.     

Mass spectrometry-based glycoprofiling of biopharmaceuticals by using an automated data processing tool: SimGlycan®

The therapeutic and immunological properties of biopharmaceuticals are governed by the glycoforms contained in them. Thus, bioinformatics tools capable of performing comprehensive characterization of glycans are significantly important to the biopharma industry. The primary structural elucidation of glycans using mass spectrometry is tricky and tedious in terms of spectral interpretation. In this study, the biosimilars of a therapeutic monoclonal antibody and an Fc-fusion protein with moderate and heavy glycosylation, respectively, were employed as representative biopharmaceuticals for released glycan analysis using liquid chromatography–tandem mass spectrometry instead of conventional mass spectrometry-based analysis. SimGlycan® is a software with proven ability to process tandem MS data for released glycans could identify eight additional glycoforms in Fc-fusion protein biosimilar, which were not detected during mass spectrometry analysis of released glycans or glyco-peptide mapping of the same molecule. Thus, liquid chromatography–tandem mass spectrometry analysis of released glycans not only complements conventional liquid chromatography–mass spectrometry-based glycan profiling but can also identify additional glycan structures that may otherwise be omitted during conventional liquid chromatography–tandem mass spectrometry based analysis of mAbs. The mass spectrometry data processing tools, such as PMI Byos™, SimGlycan^®, etc., can display pivotal analytical capabilities in automated liquid chromatography–mass spectrometry and liquid chromatography–tandem mass spectrometry-based glycan analysis workflows, especially for high-throughput structural characterization of glycoforms in biopharmaceuticals.