Fast affinity purification coupled with mass spectrometry for identifying organophosphate labeled plasma butyrylcholinesterase

Classical plasma butyrylcholinesterase (BChE) purification involves dialysis and multiple steps of chromatography. We describe a procainamide affinity gel purification scheme that takes 15-30min to purify BChE from 1ml plasma. The method uses a microfuge spin column to build a 0.2ml procainamide affinity column. The eluted BChE contains 3-4mug of 500-fold purified BChE, free from 99% of contaminating plasma proteins. The BChE was further purified by gel electrophoresis. Tryptic peptides from the BChE containing gel electrophoresis band were prepared by in-gel digestion, separated by reverse phase liquid chromatography and identified by mass spectrometry. The 29 residue active site tryptic peptide labeled with the nerve agents soman or sarin was identified.     

Rapid characterization of biotherapeutic proteins by size-exclusion chromatography coupled to native mass spectrometry

High-molecular weight aggregates such as antibody dimers and other side products derived from incorrect light or heavy chain association typically represent critical product-related impurities for bispecific antibody formats.

In this study, an approach employing ultra-pressure liquid chromatography size-exclusion separation combined with native electrospray ionization mass spectrometry for the simultaneous formation, identification and quantification of size variants in recombinant antibodies was developed. Samples exposed to storage and elevated temperature(s) enabled the identification of various bispecific antibody size variants. This test system hence allowed us to study the variants formed during formulation and bio-process development, and can thus be transferred to quality control units for routine in-process control and release analytics. In addition, native SEC-UV/MS not only facilitates the detailed analysis of low-abundant and non-covalent size variants during process characterization/validation studies, but is also essential for the SEC-UV method validation prior to admission to the market.     

Fast photochemical oxidation of proteins for comparing solvent-accessibility changes accompanying protein folding: Data processing and application to barstar

Mass spectrometry-based protein footprinting reveals regional and even amino-acid structural changes and fills the gap for many proteins and protein interactions that cannot be studied by X-ray crystallography or NMR spectroscopy. Hydroxyl radical-mediated labeling has proven to be particularly informative in this pursuit because many solvent-accessible residues can be labeled by OH in a protein or protein complex, thus providing more coverage than does specific amino-acid modifications. Finding all the OH-labeling sites requires LC/MS/MS analysis of a proteolyzed sample, but data processing is daunting without the help of automated software. We describe here a systematic means for achieving a comprehensive residue-resolved analysis of footprinting data in an efficient manner, utilizing software common to proteomics core laboratories. To demonstrate the method and the utility of OH-mediated labeling, we show that FPOP easily distinguishes the buried and exposed residues of barstar in its folded and unfolded states. This article is part of a Special Issue entitled: Mass spectrometry in structural biology.     

Chip-based separation devices coupled to mass spectrometry

The hyphenation of miniaturized separation techniques like chip electrophoresis or chip chromatography to mass spectrometry (MS) is a highly active research area in modern separation science. Such methods are particularly attractive for comprehensive analysis of complex biological samples. They can handle extremely low sample amounts, with low solvent consumption. Furthermore they provide unsurpassed analysis speed together with the prospect of integrating several functional elements on a single multifunctional platform. In this article we review the latest developments in this emerging field of technology and summarize recent trends to face current and future challenges in chip-based biochemical analysis.     

Structural mass spectrometry of membrane proteins

The analysis of proteins and protein complexes by mass spectrometry (MS) has come a long way since the invention of electrospray ionization (ESI) in the mid 80s. Originally used to characterize small soluble polypeptide chains, MS has progressively evolved over the past 3 decades towards the analysis of samples of ever increasing heterogeneity and complexity, while the instruments have become more and more sensitive and resolutive. The proofs of concepts and first examples of most structural MS methods appeared in the early 90s. However, their application to membrane proteins, key targets in the biopharma industry, is more recent. Nowadays, a wealth of information can be gathered from such MS-based methods, on all aspects of membrane protein structure: sequencing (and more precisely proteoform characterization), but also stoichiometry, non-covalent ligand binding (metals, drug, lipids, carbohydrates), conformations, dynamics and distance restraints for modelling. In this review, we present the concept and some historical and more recent applications on membrane proteins, for the major structural MS methods.     

Modifications generated by fast photochemical oxidation of proteins reflect the native conformations of proteins

Hydroxyl radical footprinting (HRF) is a nonspecific protein footprinting method that has been increasingly used in recent years to analyze protein structure. The method oxidatively modifies solvent accessible sites in proteins, which changes upon alterations in the protein, such as ligand binding or a change in conformation. For HRF to provide accurate structural information, the method must probe the native structure of proteins. This requires careful experimental controls since an abundance of oxidative modifications can induce protein unfolding. Fast photochemical oxidation of proteins (FPOP) is a HRF method that generates hydroxyl radicals via photo‐dissociation of hydrogen peroxide using an excimer laser. The addition of a radical scavenger to the FPOP reaction reduces the lifetime of the radical, limiting the levels of protein oxidation. A direct assay is needed to ensure FPOP is probing the native conformation of the protein. Here, we report using enzymatic activity as a direct assay to validate that FPOP is probing the native structure of proteins. By measuring the catalytic activity of lysozyme and invertase after FPOP modification, we demonstrate that FPOP does not induce protein unfolding.     

A method for analyzing lipid-modified proteins with mass spectrometry

In protein analysis using mass spectrometry, proteins are usually separated by electrophoresis and digested within the gel with proteases such as trypsin. However, analysis of lipid-modified proteins is difficult due to the low recovery of lipid-modified peptide fragments from the gel as well as their low ionization efficiency during mass spectrometry. In this study, we developed a simple extraction method with n-dodecyl-β-d-maltoside following chloroform/methanol extraction that efficiently elutes lipid-modified fragments from gels. This method allowed us to analyze the structure of lipid-modified fragments, suggesting the applicability of the method for analysis of lipid-modified fragments by mass spectrometry.     

Fast atom bombardment mass spectrometry of bovine proinsulin

The application of Fast Atom Bombardment Mass Spectrometry to the measurement of small differences in molecular weight is discussed in relation to the spectra of bovine proinsulin and a genetic variant.     

Fast atom bombardment mass spectrometry of human proinsulin

The observation of protonated molecular species from human proinsulin obtained by fast atom bombardment mass spectrometry is reported.     

Shotgun glycopeptide capture approach coupled with mass spectrometry for comprehensive glycoproteomics

We present a robust and general shotgun glycoproteomics approach to comprehensively profile glycoproteins in complex biological mixtures. In this approach, glycopeptides derived from glycoproteins are enriched by selective capture onto a solid support using hydrazide chemistry followed by enzymatic release of the peptides and subsequent analysis by tandem mass spectrometry. The approach was validated using standard protein mixtures that resulted in a close to 100% capture efficiency. Our capture approach was then applied to microsomal fractions of the cisplatin-resistant ovarian cancer cell line IGROV-1/CP. With a Protein Prophet probability value greater than 0.9, we identified a total of 302 proteins with an average protein identification rate of 136 +/- 19 (n = 4) in a single linear quadrupole ion trap (LTQ) mass spectrometer nano-LC-MS experiment and a selectivity of 91 +/- 1.6% (n = 4) for the N-linked glycoconsensus sequence. Our method has several advantages. 1) Digestion of proteins initially into peptides improves the solubility of large membrane proteins and exposes all of the glycosylation sites to ensure equal accessibility to capture reagents. 2) Capturing glycosylated peptides can effectively reduce sample complexity and at the same time increase the confidence of MS-based protein identifications (more potential peptide identifications per protein). 3) The utility of sodium sulfite as a quencher in our capture approach to replace the solid phase extraction step in an earlier glycoprotein chemical capture approach for removing excess sodium periodate allows the overall capture procedure to be completed in a single vessel. This improvement minimizes sample loss, increases sensitivity, and makes our protocol amenable for high throughput implementation, a feature that is essential for biomarker identification and validation of a large number of clinical samples. 4) The approach is demonstrated here on the analysis of N-linked glycopeptides; however, it can be applied equally well to O-glycoprotein analysis.     

Use of stable isotopically enriched proteins and directly coupled high-performance liquid chromatography inductively coupled plasma mass spectrometry for quantitatively monitoring the transfer of metals between proteins

Studies have shown that metallothionein (MT) may play an important role in modulating the activity of certain Zn-regulated enzymes under various oxidoreductive conditions by either donating or removing Zn. To better determine the role of MT in interprotein metal transfer, we describe a procedure that uses stable isotopically enriched (67)Zn(7) metallothionein 2 ((67)Zn(7)-MT-2) to quantitatively determine the stoichiometry of transfer of Zn from the protein to a recipient apo-metalloenzyme, apo-carbonic anhydrase (apo-CA) by directly coupled ion exchange high-performance liquid chromatography inductively coupled plasma mass spectrometry. Quantitatively, the transfer of (67)Zn was consistent with the enzymatic activation of the apo-enzyme as judged by its esterase activity and ability to cleave p-nitrophenyl acetate. Maximum enzyme activation occurred at an MT-2:apo-CA molar ratio of 1, implying the release of a single atom of Zn from MT-2. Preincubation of (67)Zn(7)-MT-2 with an excess of oxidized glutathione (GSSG) increased metal donation fourfold, whereas reduced glutathione (GSH) inhibited donation by approximately 50%. By using multiple recipient and donor proteins having different stable isotopic signatures, the technique has the potential for quantitatively studying the kinetic and thermodynamic aspects of Zn transfer between numerous competing ligands in vitro, an important first step toward understanding the regulatory role of this metal in protein functioning and cellular metabolism in vivo.     

Fast multi-blind modification search through tandem mass spectrometry

With great biological interest in post-translational modifications (PTMs), various approaches have been introduced to identify PTMs using MS/MS. Recent developments for PTM identification have focused on an unrestrictive approach that searches MS/MS spectra for all known and possibly even unknown types of PTMs at once. However, the resulting expanded search space requires much longer search time and also increases the number of false positives (incorrect identifications) and false negatives (missed true identifications), thus creating a bottleneck in high throughput analysis. Here we introduce MODa, a novel "multi-blind" spectral alignment algorithm that allows for fast unrestrictive PTM searches with no limitation on the number of modifications per peptide while featuring over an order of magnitude speedup in relation to existing approaches. We demonstrate the sensitivity of MODa on human shotgun proteomics data where it reveals multiple mutations, a wide range of modifications (including glycosylation), and evidence for several putative novel modifications. Based on the reported findings, we argue that the efficiency and sensitivity of MODa make it the first unrestrictive search tool with the potential to fully replace conventional restrictive identification of proteomics mass spectrometry data.     

Fast atom bombardment mass spectrometry analysis of opioid peptides

Positive and negative ion fast atom bombardment mass spectrometries have been used to determine the amino acid sequence-determining fragment ion information of opioid peptides containing from 5 to 10 amino acid residues. The opioids investigated include several enkephalins, dynorphin A fragments 1-7 through 1-10, and alpha- and beta-neoendorphins. Data obtained in the two ionization polarities provide complementary information and exhibit the C-terminal- and the N-terminal-containing amino acid sequence-determining fragment ions that are formed by cleavage of the bond between the carbonyl group and the alpha-carbon (-CHR-CO-), the peptide amide bond (-CO-NH-), and the amino-alkyl (-NH-CHR-) bond. The C-terminal sequence ions are dominant in the positive ion mode, whereas the C-terminal and N-terminal ions are equally important in the negative ion mode. Detection limits for full mass scans extend down to the picomole range. The apparent role of hydrophobicity of the amino acid residues on the fragmentation characteristics of the peptide is discussed.     

Fast atom bombardment mass spectrometry of metal nucleotide complexes

The fast atom bombardment mass spectrometry of the beta-gamma complexes of ATP and GTP with cobalt and chromium are reported. Spectra were recorded in the positive ion mode. Ions in the molecular weight region allow identification of the complexes to be made.     

Functional assessment of antibody oxidation by native mass spectrometry

Oxidation of methionine (Met) residues is one of several chemical degradation pathways for recombinant IgG1 antibodies. Studies using several methodologies have indicated that Met oxidation in the constant IgG1 domains affects in vitro interaction with human neonatal Fc (huFcRn) receptor, which is important for antibody half-life. Here, a completely new approach to investigating the effect of oxidative stress conditions has been applied. Quantitative ultra-performance liquid chromatography mass spectrometry (MS) peptide mapping, classical surface plasmon resonance and the recently developed FcRn column chromatography were combined with the new fast-growing approach of native MS as a near native state protein complex analysis in solution. Optimized mass spectrometric voltage and pressure conditions were applied to stabilize antibody/huFcRn receptor complexes in the gas phase for subsequent native MS experiments with oxidized IgG1 material. This approach demonstrated a linear correlation between quantitative native MS and IgG-FcRn functional analysis.

In our study, oxidation of the heavy chain Met-265 resulted in a stepwise reduction of mAb3/huFcRn receptor complex formation. Remarkably, a quantitative effect of the heavy chain Met-265 oxidation on relative binding capacity was only detected for doubly oxidized IgG1, whereas IgG1 with only one oxidized heavy chain Met-265 was not found to significantly affect IgG1 binding to huFcRn. Thus, mono-oxidized IgG1 heavy chain Met-265 most likely does not represent a critical quality attribute for pharmacokinetics.