Top-down MS Research Articles

Top-down mass spectrometry of cardiac myofilament proteins in health and disease.

Myofilaments are composed of thin and thick filaments that coordinate with each other to regulate muscle contraction and relaxation. PTMs together with genetic variations and alternative splicing of the myofilament proteins play essential roles in regulating cardiac contractility in health and disease. Therefore, a comprehensive characterization of the myofilament proteins in physiological and pathological conditions is essential for better understanding the molecular basis of cardiac function and dysfunction. Due to the vast complexity and dynamic nature of proteins, it is challenging to obtain a holistic view of myofilament protein modifications. In recent years, top-down MS has emerged as a powerful approach to study isoform composition and PTMs of proteins owing to its advantage of complete sequence coverage and its ability to identify PTMs and sequence variants without a priori knowledge. In this review, we will discuss the application of top-down MS to the study of cardiac myofilaments and highlight the insights it provides into the understanding of molecular mechanisms in contractile dysfunction of heart failure. Particularly, recent results of cardiac troponin and tropomyosin modifications will be elaborated. The limitations and perspectives on the use of top-down MS for myofilament protein characterization will also be briefly discussed.

Ion mobility and Top-down MS complementary approaches for the structural analysis of protein models bound to anticancer metallodrugs

Interactions between proteins and platinum (Pt) compounds lie at the heart of both the effectiveness of Pt-based drugs and their side effects. A greater understanding of such interactions at the molecular level would contribute to the design of Pt compounds for future clinical use. In this regard, here we used ion mobility mass spectrometry (IM-MS) to study the structural behavior of three dicarboxylate platinum compounds, namely [Ptcbdca(dach)] (trans-1,2-cyclohexanediamine)(1,1-cyclobutanedicarbo-xylato)platinum(II), [Pt(4-hmpy)2mdbs]bis[(4-pyridinemethanol)(meso-2,3-dibromosuccinato)]platinum(II) and [Ptdcch(4-hmpy)2]bis-[(4-pyridinemethanol)(trans-1,2-cyclo-hexanedicarboxylato)]platinum(II), upon binding to model proteins involved in carcinogenic or anti-carcinogenic processes (Ubiquitin, Cytochrome C and potato carboxypeptidase inhibitor (PCI)). We found that the three Pt compounds bound Ubiquitin, PCI and Cytochrome C, highlighting the formation of biadducts and triadducts with the latter. Using a competitive assay with glutathione, we also explored the potential transfer of Pt to biological nucleophiles and particularly to DNA in protein-containing methionine-Pt adducts. HPLC-MS [ESI(+)] studies demonstrated the capacity of glutathione to scavenge the Pt attached to the protein moiety. Furthermore, using targeted Top-down mass spectrometry, we identified N-terminal Met1 and C-terminal His68 residues as the preferential binding of the dicarboxylate platinum compounds sites on Ubiquitin.

Qualitative and quantitative peptidomic and proteomic approaches to phenotyping chicken semen.

This peptidomic and proteomic study i) characterized for the first time the semen protein composition of the main domestic avian species (Gallus gallus) by analysis of ejaculated spermatozoa and corresponding seminal plasma; ii) established a characteristic molecular phenotype distinguishing semen and males at an individual level; and iii) proposedthe first evidence of biomarkers related to fertility.

Investigating macromolecular complexes using top-down mass spectrometry.

MS has emerged as an important tool to investigate noncovalent interactions between proteins and various ligands (e.g. other proteins, small molecules, or drugs). In particular, ESI under so-called "native conditions" (a.k.a. "native MS") has considerably expanded the scope of such investigations. For instance, ESI quadrupole time of flight (Q-TOF) instruments have been used to probe the precise stoichiometry of protein assemblies, the interactions between subunits and the position of subunits within the complex (i.e. defining core and peripheral subunits). This review highlights several illustrative native Q-TOF-based investigations and recent seminal contributions of top-down MS (i.e. Fourier transform (FT) MS) to the characterization of noncovalent complexes. Combined top-down and native MS, recently demonstrated in "high-mass modified" orbitrap mass spectrometers, and further improvements needed for the enhanced investigation of biologically significant noncovalent interactions by MS will be discussed.

Ultraviolet photodissociation enhances top-down mass spectrometry as demonstrated on green fluorescent protein variants.

Ultraviolet photodissociation (UVPD) is a compelling fragmentation technique with great potential to enhance proteomics generally and top-down MS specifically. In this issue, Cannon et al. (Proteomics 2014, 14, XXXX-XXXX) use UVPD to perform top-down MS on several sequence variants of green fluorescent protein and compare the results to CID, higher energy collision induced dissociation, and electron transfer dissociation. As compared to the other techniques UVPD produces a wider variety of fragment ion types that are relatively evenly distributed across the protein sequences. Overall, their results demonstrate enhanced sequence coverage and higher confidence in sequence assignment via UVPD MS. Based on these and other recent results UVPD is certain to become an increasingly widespread and valuable tool for top-down proteomics.

Determination of disulfide linkages in antimicrobial peptides of the macin family by combination of top-down and bottom-up proteomics.

Beside the high biological significance which is based on the inherent dependence of biological activity on the structural features of antimicrobial peptides (which holds true for entirely every protein), the presented analytical strategy will be of wide interest, as it widens the available toolbox for the analysis of this important posttranslational modification.

The first pilot project of the consortium for top‐down proteomics: A status report

Pilot Project #1--the identification and characterization of human histone H4 proteoforms by top-down MS--is the first project launched by the Consortium for Top-Down Proteomics (CTDP) to refine and validate top-down MS. Within the initial results from seven participating laboratories, all reported the probability-based identification of human histone H4 (UniProt accession P62805) with expectation values ranging from 10(-13) to 10(-105). Regarding characterization, a total of 74 proteoforms were reported, with 21 done so unambiguously; one new PTM, K79ac, was identified. Inter-laboratory comparison reveals aspects of the results that are consistent, such as the localization of individual PTMs and binary combinations, while other aspects are more variable, such as the accurate characterization of low-abundance proteoforms harboring >2 PTMs. An open-access tool and discussion of proteoform scoring are included, along with a description of general challenges that lie ahead including improved proteoform separations prior to mass spectrometric analysis, better instrumentation performance, and software development.

An integrated top‐down and bottom‐up proteomic approach to characterize the antigen‐binding fragment of antibodies

We have previously shown that different individuals exposed to the same antigen produce antibodies with identical mutations in their complementarity determining regions (CDR), suggesting that CDR tryptic peptides can serve as biomarkers for disease diagnosis and prognosis. Complete Fabs derived from disease specific antibodies have even higher potential; they could potentially be used for disease treatment and are required to identify the antigens toward which the antibodies are directed. However, complete Fab sequence characterization via LC-MS analysis of tryptic peptides (i.e. bottom-up) has proven to be impractical for mixtures of antibodies. To tackle this challenge, we have developed an integrated bottom-up and top-down MS approach, employing 2D chromatography coupled with Fourier transform mass spectrometry (FTMS), and applied this approach for full characterization of the variable parts of two pharmaceutical monoclonal antibodies with sensitivity comparable to the bottom-up standard. These efforts represent an essential step toward the identification of disease specific antibodies in patient samples with potentially significant clinical impact.

Top‐down mass spectrometry and hydrogen/deuterium exchange for comprehensive structural characterization of interferons: Implications for biosimilars

Rapid development in biopharmaceuticals has put high demands on analytical tools that can provide accurate and comprehensive characterization of protein drugs, including biosimilars. Although the enzyme digestion based "bottom-up" approach is usually the method of choice for this purpose, it only gives peptide-level information and sequence coverage is often incomplete. In this work, we used top-down MS with electron capture dissociation (ECD) to characterize both the primary and higher order structures of a therapeutic protein interferon and its variants. Accurate mass measurement at the intact protein level combined with top-down ECD fragmentation enabled unambiguous protein sequence confirmation and identification of all PTMs. Combining hydrogen/deuterium exchange and rapid disulfide reduction with top-down ECD on the LC time scale, we have investigated the differences in higher order structure between the protein variants, as well as the impact of PTMs on protein conformation.

Top-Down Proteomics with Mass Spectrometry Imaging: A Pilot Study towards Discovery of Biomarkers for Neurodevelopmental Disorders

In the developing mammalian brain, inhibition of NMDA receptor can induce widespread neuroapoptosis, inhibit neurogenesis and cause impairment of learning and memory. Although some mechanistic insights into adverse neurological actions of these NMDA receptor antagonists exist, our understanding of the full spectrum of developmental events affected by early exposure to these chemical agents in the brain is still limited. Here we attempt to gain insights into the impact of pharmacologically induced excitatory/inhibitory imbalance in infancy on the brain proteome using mass spectrometric imaging (MSI). Our goal was to study changes in protein expression in postnatal day 10 (P10) rat brains following neonatal exposure to the NMDA receptor antagonist dizocilpine (MK801). Analysis of rat brains exposed to vehicle or MK801 and comparison of their MALDI MS images revealed differential relative abundances of several proteins. We then identified these markers such as ubiquitin, purkinje cell protein 4 (PEP-19), cytochrome c oxidase subunits and calmodulin, by a combination of reversed-phase (RP) HPLC fractionation and top-down tandem MS platform. More in-depth large scale study along with validation experiments will be carried out in the future. Overall, our findings indicate that a brief neonatal exposure to a compound that alters excitatory/inhibitory balance in the brain has a long term effect on protein expression patterns during subsequent development, highlighting the utility of MALDI-MSI as a discovery tool for potential biomarkers.

Top‐down protein identification of proteasome proteins with nanoLC‐FT‐ICR‐MS employing data‐independent fragmentation methods

A comparison of different data-independent fragmentation methods combined with LC coupled to high-resolution FT-ICR-MS/MS is presented for top-down MS of protein mixtures. Proteins composing the 20S and 19S proteasome complexes and their PTMs were identified using a 15 T FT-ICR mass spectrometer. The data-independent fragmentation modes with LC timescales allowed for higher duty-cycle measurements that better suit online LC-FT-ICR-MS. Protein top-down dissociation was effected by funnel-skimmer collisionally activated dissociation (FS-CAD) and CASI (continuous accumulation of selected ions)-CAD. The N-termini for 9 of the 14 20S proteasome proteins were found to be modified, and the α3 protein was found to be phosphorylated; these results are consistent with previous reports. Mass-measurement accuracy with the LC-FT-ICR system for the 20- to 30-kDa 20S proteasome proteins was 1 ppm. The intact mass of the 100-kDa Rpn1 subunit from the 19S proteasome complex regulatory particle was measured with a deviation of 17 ppm. The CASI-CAD technique is a complementary tool for intact-protein fragmentation and is an effective addition to the growing inventory of dissociation methods that are compatible with online protein separation coupled to FT-ICR-MS.

Qualitative and quantitative top‐down mass spectral analysis of crustacean hyperglycemic hormones in response to feeding

An efficient pipeline for peptide discovery accelerates peptidomic analysis and facilitates a better understanding of the functional roles of neuropeptides. However, qualitative and quantitative analysis of large neuropeptides is challenging due to the bigger molecular sizes, multiple PTMs, and interference by homologous isoforms. Herein, we refined two methodologies in the pipeline for highly confident and efficient MS-based peptide discovery. For the qualitative analysis, the so-called "high resolution/accurate mass" measurement on Orbitrap mass spectrometers was integrated with computer-assisted homology search, which was successfully applied to decipher the substituted amino acid residues in large neuropeptides by referring to homologous sequences. For the quantitative analysis, a new isotopic labeling-assisted top-down MS strategy was developed, which enabled direct monitoring of the abundance changes of endogenous large neuropeptides. By using the refined peptide discovery pipeline, one novel crustacean hyperglycemic hormone (CHH) from the Dungeness crab sinus glands was confidently identified and de novo sequenced, and its relative abundance was quantified. Comparative analysis of CHHs in unfed and fed crabs revealed that the peptide abundance in the sinus glands was significantly increased after food intake, suggesting that the release of CHHs might be altered by feeding behavior.

Complete posttranslational modification mapping of pathogenic Neisseria meningitidis pilins requires top‐down mass spectrometry

In pathogenic bacteria, posttranslationally modified proteins have been found to promote bacterial survival, replication, and evasion from the host immune system. In the human pathogen Neisseria meningitidis, the protein PilE (15-18 kDa) is the major building block of type IV pili, extracellular filamentous organelles that play a major role in mediating pathogenesis. Previous reports have shown that PilE can be expressed as a number of different proteoforms, each harboring its own set of PTMs and that specific proteoforms are key in promoting bacterial virulence. Efficient tools that allow complete PTM mapping of proteins involved in bacterial infection are therefore strongly needed. As we show in this study, a simple combination of mass profiling and bottom-up proteomics is fundamentally unable to achieve this goal when more than two proteoforms are present simultaneously. In a N. meningitidis strain isolated from a patient with meningitis, mass profiling revealed the presence of four major proteoforms of PilE, in a 1:1:1:1 ratio. Due to the complexity of the sample, a top-down approach was required to achieve complete PTM mapping for all four proteoforms, highlighting an unprecedented extent of glycosylation. Top-down MS therefore appears to be a promising tool for the analysis of highly posttranslationally modified proteins involved in bacterial virulence.

Compound heterozygotes and beta‐thalassemia: Top‐down mass spectrometry for detection of hemoglobinopathies

We have shown previously that liquid extraction surface analysis of dried blood spots coupled to high resolution top-down MS may be applied for the diagnosis of hemoglobin (Hb) variants FS, FAS, FC, FAC, FAD in newborn samples. The objective of the current work was to determine whether the structural variant HbE, compound heterozygote variants FSC and FSD, and β-thalassemia were amenable to diagnosis by this approach. Anonymized residual neonatal dried blood spot samples, taken as part of the routine newborn screening program, were analyzed by liquid extraction surface analysis coupled to high resolution MS/MS. The samples had been previously screened and were known to be FAE, FSC, FSD, or β-thalassemia. Manual analysis of the mass spectra revealed that, in all cases, the variants may be confirmed. Direct surface sampling MS should be considered as an alternative to current screening techniques for the diagnosis of Hb variants.

Using Mass Spectrometry to Monitor Monoclonal Immunoglobulins in Patients with a Monoclonal Gammopathy

A monoclonal gammopathy is defined by the detection a monoclonal immunoglobulin (M-protein). In clinical practice, the M-protein is detected by protein gel electrophoresis (PEL) and immunofixation electrophoresis (IFE). We theorized that molecular mass could be used instead of electrophoretic patterns to identify and quantify the M-protein because each light and heavy chain has a unique amino acid sequence and thus a unique molecular mass whose increased concentration could be distinguished from the normal polyclonal background. In addition, we surmised that top-down MS could be used to isotype the M-protein because each immunoglobulin has a constant region with an amino acid sequence unique to each isotype. Our method first enriches serum for immunoglobulins followed by reduction using DTT to separate light chains from heavy chains and then by microflow LC-ESI-Q-TOF MS. The multiply charged light and heavy chain ions are converted to their molecular masses, and reconstructed peak area calculations for light chains are used for quantification. Using this method, we demonstrate how the light chain portion of an M-protein can be monitored by molecular mass, and we also show that in sequential samples from a patient with multiple myeloma the light chain portion of the M-protein was detected in all samples, even those negative by PEL, IFE, and quantitative FLC. We also present top-down MS isotyping of M-protein light chains using a unique isotype-specific fragmentation pattern allowing for quantification and isotype identification in the same run. Our results show that microLC-ESI-Q-TOF MS provides superior sensitivity and specificity compared to conventional methods and shows promise as a viable method of detecting and isotyping an M-protein.

Identification and discrimination of binding sites of an organoruthenium anticancer complex to single-stranded oligonucleotides by mass spectrometry.

We here report the identification of the binding sites of an organometallic ruthenium anticancer complex [(η(6)-biphenyl)Ru(en)Cl](+) (1) to single-stranded oligodeoxynucleotides (ODNs) 5'-CCCA4G5C6CC-3' (I) and 5'-CCC3G4A5CCC-3' (II) by mass spectrometry. The MS analysis of exonuclease ladders demonstrated that the 5'-exonuclease bovine spleen phosphodiesterase digestion of I and II mono-ruthenated by complex 1 was arrested solely at A4 and partially at C3 and G4, respectively, and that the 3'-exonuclease snake venom phosphodiesterase digestion of the ruthenated ODNs was arrested solely at G5 and G4, respectively, due to the ruthenation. These results did not allow unambiguous identification of ruthenation sites on the metallated ODNs. In contrast, tandem mass spectrometry analysis with CID fragmentation of the mono-ruthenated ODNs provided sequential and complementary [a(i) - B]/wi fragments, leading to unambiguous identification of G5 in I and G4 in II as the ruthenation sites on the ODN adducts, which is in line with the high selectivity of the complex towards guanine base as reported previously. These findings suggest that caution should be raised with regard to the identification of the binding sites of metal complexes, in particular complexes with bulky ligands, like biphenyl in complex 1, to DNA by MS analysis of exonuclease ladders of the metallated adducts, because the bulky ligands may adopt such an orientation that they block the exonuclease cleavage of the 5'- or 3'-side phosphodiester bonds adjacent to the binding sites, leading to digestion stalling at the nucleotides before the binding sites.

The top‐down, middle‐down, and bottom‐up mass spectrometry approaches for characterization of histone variants and their post‐translational modifications

Epigenetic regulation of gene expression is, at least in part, mediated by histone modifications. PTMs of histones change chromatin structure and regulate gene transcription, DNA damage repair, and DNA replication. Thus, studying histone variants and their modifications not only elucidates their functional mechanisms in chromatin regulation, but also provides insights into phenotypes and diseases. A challenge in this field is to determine the best approach(es) to identify histone variants and their PTMs using a robust high-throughput analysis. The large number of histone variants and the enormous diversity that can be generated through combinatorial modifications, also known as histone code, makes identification of histone PTMs a laborious task. MS has been proven to be a powerful tool in this regard. Here, we focus on bottom-up, middle-down, and top-down MS approaches, including CID and electron-capture dissociation/electron-transfer dissociation based techniques for characterization of histones and their PTMs. In addition, we discuss advances in chromatographic separation that take advantage of the chemical properties of the specific histone modifications. This review is also unique in its discussion of current bioinformatic strategies for comprehensive histone code analysis.

Native top-down electrospray ionization-mass spectrometry of 158 kDa protein complex by high-resolution Fourier transform ion cyclotron resonance mass spectrometry.

Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) delivers high resolving power, mass measurement accuracy, and the capabilities for unambiguously sequencing by a top-down MS approach. Here, we report isotopic resolution of a 158 kDa protein complex, tetrameric aldolase with an average absolute deviation of 0.36 ppm and an average resolving power of ~520 000 at m/z 6033 for the 26+ charge state in magnitude mode. Phase correction further improves the resolving power and average absolute deviation by 1.3-fold. Furthermore, native top-down electron capture dissociation (ECD) enables the sequencing of 168 C-terminal amino acid (AA) residues out of 463 total AAs. Combining the data from top-down MS of native and denatured aldolase complexes, a total of 56% of the total backbone bonds were cleaved. The observation of complementary product ion pairs confirms the correctness of the sequence and also the accuracy of the mass fitting of the isotopic distribution of the aldolase tetramer. Top-down MS of the native protein provides complementary sequence information to top-down ECD and collisonally activated dissociation (CAD) MS of the denatured protein. Moreover, native top-down ECD of aldolase tetramer reveals that ECD fragmentation is not limited only to the flexible regions of protein complexes and that regions located on the surface topology are prone to ECD cleavage.

Unequivocal Determination of Site-Specific Protein Disulfide Bond Reduction Potentials by Top-Down FTICR MS: Characterization of the N- and C-Terminal Redox-Active Sites in Human Thioredoxin 1

We report the reliable determination of equilibrium protein disulfide bond reduction potentials (E°') by isotope-coded cysteine alkylation coupled with top-down Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS). This technique enables multiple redox-active sites to be characterized simultaneously and unambiguously without the need for proteolysis or site-directed mutagenesis. Our model system was E. coli thioredoxin, and we determined E°' for its CGPC active-site disulfide as -280 mV in accord with literature values. E°' for the homologous disulfide in human thioredoxin 1 (Trx1) was determined as -281 mV, a value considerably more negative than the previously reported -230 mV. We also observed S-glutathionylation of Trx1 and localized that redox modification to Cys72; E°' for the intermolecular disulfide was determined as -186 mV. Intriguingly, that value corresponds to the intracellular glutathione/glutathione disulfide (GSH/GSSG) potential at the redox boundary between cellular differentiation and apoptosis.

Ultrahigh pressure fast size exclusion chromatography for top-down proteomics

Top-down MS-based proteomics has gained a solid growth over the past few years but still faces significant challenges in the LC separation of intact proteins. In top-down proteomics, it is essential to separate the high mass proteins from the low mass species due to the exponential decay in S/N as a function of increasing molecular mass. SEC is a favored LC method for size-based separation of proteins but suffers from notoriously low resolution and detrimental dilution. Herein, we reported the use of ultrahigh pressure (UHP) SEC for rapid and high-resolution separation of intact proteins for top-down proteomics. Fast separation of intact proteins (6-669 kDa) was achieved in < 7 min with high resolution and high efficiency. More importantly, we have shown that this UHP-SEC provides high-resolution separation of intact proteins using a MS-friendly volatile solvent system, allowing the direct top-down MS analysis of SEC-eluted proteins without an additional desalting step. Taken together, we have demonstrated that UHP-SEC is an attractive LC strategy for the size separation of proteins with great potential for top-down proteomics.