Ion soft-landing into liquids: Protein identification,separation, and purification with retention of biological activity

Protein ions, after mass spectrometric separation, can be soft-landed into liquid surfaces with preservation of their native structures. Retention of biological activity is strongly favored in glycerol-based surfaces but not in self-assembled monolayer solid surfaces. Soft-landing efficiency for multiply-charged hexokinase ions was found to be some four times higher for a glycerol/fructose liquid surface than for a fluorinated self-assembled monolayer surface. Soft-landing into liquid surfaces is also shown to allow (1) protein purification, (2) on-surface identification of the soft-landed material using MALDI, and (3) protein identification by in-surface tryptic digestion. Pure lysozyme was successfully isolated from different mixtures including an oxidized, partially decomposed batch of the protein and a partial tryptic digest. Liquid glycerol/carbohydrate mixtures could be used directly to record MALDI spectra on the soft-landed compounds provided they were fortified in advance with traditional MALDI matrices such as p-nitroaniline and alpha-cyano-4-hydroxycinnamic acid. Various proteins were soft-landed and detected on-target using these types of liquid surface. Soft-landing of multiply-charged lysozyme ions onto fluorinated self-assembled monolayer surfaces was found to occur with a limited amount of neutralization, and trapped multiply-charged ions could be desorbed from the surface by laser desorption. Initial data is shown for a new approach to protein identification that combines top-down and bottom-up approaches by utilizing protein ion soft-landing from a protein mixture, followed by tryptic digestion of the landed material and detection of characteristic tryptic fragments by MALDI.     

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.     

Disparities between immobilized enzyme and solution based digestion of transferrin with trypsin

Trypsin digestion is a major component of preparing proteins for peptide based identification and quantification by mass spectral (MS) analysis. Surprisingly proteolysis is the slowest part of the proteomics process by an order of magnitude. Numerous recent efforts to reduce protein digestion to a few minutes have centered on the use of an immobilized enzyme reactor (IMER) to minimize both trypsin autolysis and vastly increase the trypsin to protein ratio. A central question in this approach is whether proteolysis with an IMER produces the same peptide cleavage products as derived from solution based digestion. The studies reported here examined this question with transferrin; a model protein of known resistance to trypsin digestion. Results from these studies confirmed that a trypsin‐IMER can in fact digest transferrin in a few minutes; providing tryptic peptides that subsequent to MS analysis allow sequence identification equivalent to solution digestion. Although many of the peptides obtained from these two trypsin digestion systems were identical, many were not. The greatest difference was that the trypsin‐ IMER produces (i) numerous peptides bearing multiple lysine and/or arginine residues and (ii) identical portions of the protein sequence were found in multiple peptides. Most of these peptides were derived from five regions in transferrin. These results were interpreted to mean that proteolysis in the case of transferrin occurred faster than the rate at which buried lysine and arginine residues were unmasked in the five regions providing peptides that were only partially digested.     

Integration of capillary isoelectric focusing with monolithic immobilized pH gradient,immobilized trypsin microreactor and capillary zone electrophoresis for on‐line protein analysis

An integrated platform consisting of protein separation by CIEF with monolithic immobilized pH gradient (M‐IPG), on‐line digestion by trypsin‐based immobilized enzyme microreactor (trypsin‐IMER), and peptide separation by CZE was established. In such a platform, a tee unit was used not only to connect M‐IPG CIEF column and trypsin‐IMER, but also to supply adjustment buffer to improve the compatibility of protein separation and digestion. Another interface was made by a Teflon tube with a nick to couple IMER and CZE via a short capillary, which was immerged in a centrifuge tube filled with 20 mmol/L glutamic acid, to exchange protein digests buffer and keep electric contact for peptide separation. By such a platform, under the optimal conditions, a mixture of ribonuclease A, myoglobin and BSA was separated into 12 fractions by M‐IPG CIEF, followed by on‐line digestion by trypsin‐IMER and peptide separation by CZE. Many peaks of tryptic peptides, corresponding to different proteins, were observed with high UV signals, indicating the excellent performance of such an integrated system. We hope that the CE‐based on‐line platform developed herein would provide another powerful alternative for an integrated analysis of proteins.     

Implications of partial tryptic digestion in organic-aqueous solvent systems for bottom-up proteome analysis

For bottom-up MS, the digestion step is critical and is typically performed with trypsin. Solvent-assisted digestion in 80% acetonitrile has previously been shown to improve protein sequence coverage at shorter digestion times. This has been attributed to enhanced enzyme digestion efficiency in this solvent. However, our results demonstrate that tryptic digestion in 80% acetonitrile is less efficient than that of conventional (aqueous) digestion. This is a consequence of decreased enzyme activity beyond ∼40% acetonitrile, increased enzyme autolysis and lower protein solubility in 80% acetonitrile. We observe multiple missed cleavages and reduced concentration of fully cleaved digestion products. Nonetheless we confirm, through room temperature solvent-assisted digestion, a consistent improvement in protein sequence coverage when analyzed by mass spectrometry. These results are explained through the increased number of unique digestion products available for detection. Thus, while solvent-assisted digestion has clear merits for proteome analysis, one should be aware of the inefficiency of protein digestion though this protocol, particularly with absolute protein quantitation experiments.     

High throughput tryptic digestion via poly (acrylamide-co-methylenebisacrylamide) monolith based immobilized enzyme reactor

A poly (acrylamide-co-methylenebisacrylamide) (poly (AAm-co-MBA)) monolith was prepared by thermal polymerization in the 100 or 250 μm i.d. capillary. The monolithic support was activated by ethylenediamine followed by glutaraldehyde. Trypsin was then introduced to form an immobilized enzyme reactor (IMER). The prepared IMER showed a reliable mechanical stability and permeability (permeability constant K = 2.65 × 10⁻¹³ m²). With BSA as the model protein, efficient digestion was completed within 20 s, yielding the sequence coverage of 57%, better than that obtained from the traditional in-solution digestion (42%), which took about 12 h. Moreover, BSA down to femtomole was efficiently digested by the IMER and positively identified by matrix assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). To test the applicability of IMER for complex sample profiling, proteins extracted from Escherichia coli were digested by the IMER and further analyzed by nanoreversed phase liquid chromatography-electrospray ionization-mass spectrometry (nanoRPLC-ESI-MS/MS). In comparison to in-solution digestion, despite slightly fewer proteins were positively identified at a false discovery rate (FDR) of ∼1% (333 vs 411), the digestion time used was largely shortened (20 s vs 24 h), implying superior digestion performance for the high throughput analysis of complex samples.     

Determination of the binding sites for oxaliplatin on insulin using mass spectrometry-based approaches

Using insulin as a model protein for binding of oxaliplatin to proteins, various mass spectrometric approaches and techniques were compared. Several different platinum adducts were observed, e.g. addition of one or two diaminocyclohexane platinum(II) (Pt(dach)) molecules. By top-down analysis and fragmentation of the intact insulin–oxaliplatin adduct using nano-electrospray ionisation quadrupole time-of-flight mass spectrometry (nESI-Q-ToF-MS), the major binding site was assigned to histidine5 on the insulin B chain. In order to simplify the interpretation of the mass spectrum, the disulphide bridges were reduced. This led to the additional identification of cysteine6 on the A chain as a binding site along with histidine5 on the B chain. Digestion of insulin–oxaliplatin with endoproteinase Glu-C (GluC) followed by reduction led to the formation of five peptides with Pt(dach) attached. Identification of several of the binding sites was obtained using matrix-assisted laser desorption/ionization (MALDI)-ToF-ToF-MS and liquid chromatography-nESI-Q-ToF-MS. Upon comparing the top-down and bottom-up approaches, the suitability of the bottom-up approach for determining binding sites was questioned, as the release and possible re-association of Pt(dach) were demonstrated upon enzymatic digestion. The associated advantages and disadvantages of ESI and MALDI were also pointed out.     

Integrated platform of capillary isoelectric focusing, trypsin immobilized enzyme microreactor and nanoreversed-phase liquid chromatography with mass spectrometry for online protein profiling

An integrated platform with the combination of protein and peptide separation was established via online protein digestion, by which proteins were first separated by CIEF, online digested by a trypsin immobilized enzyme microreactor, trapped and desalted by two parallel trap columns, separated by nanoreversed-phase and finally identified by MS. In such a platform, two hollow fiber membrane interfaces were used. One was applied to supply catholyte and electric contact, and another to supply adjustment buffer to improve the compatibility of protein separation and tryptic digestion. A poly(octadecyl acrylate-co-ethylene dimethacrylate) monolithic column served as the trap column to capture sample and to remove the ampholytes from CIEF. A hybrid silica monolith-based immobilized trypsin microreactor was used for online protein digestion. To evaluate the performance of such a platform, a 4-protein mixture with a loading amount of only 0.29?μg, was analyzed, and sequence coverages for BSA, myoglobin, β-lactoglobulin and ribonuclease A were 8, 26, 10 and 54%, respectively. Furthermore, such an integrated platform was successfully applied for the analysis of proteins extracted from Escherichia coli, and 101 proteins were positively identified. We anticipate that the integrated platform developed herein will provide a promising tool for low-abundance protein identification with the combination of top-down and bottom-up approaches.     

Efficient Proteolysis of Glycoprotein Using a Hydrophilic Immobilized Enzyme Reactor Coupled with MALDI-QIT-TOF-MS Detection and μHPLC Analysis

A hydrophilic immobilized enzyme reactor (IMER) containing trypsin was prepared and applied in the proteolysis of glycoproteins. Glycoproteins including horseradish peroxidase, asialofetuin, and fetuin were used to evaluate the performance of the hydrophilic IMER for the glycoprotein digestion. The digested products were detected by matrix-assisted laser desorption/ionization quadruple ion trap time-of-flight mass spectrometry and micro-high-performance liquid chromatography. The hydrophilic IMER showed higher enzymatic digestion efficiency compared with conventional in-solution digestion. The digestion time could be reduced from 16 h to several minutes. Furthermore, using microwaves as a heat source, the reproducibility of the hydrophilic IMER was evaluated and this IMER could be recycled for at least ten times without obvious loss of enzyme activity. The hydrophilic IMER provides a promising tool for high-throughput glycoproteome analysis.     

A multiparameter optimization in middle-down analysis of monoclonal antibodies by LC–MS/MS

In antibody-based drug research, a complete characterization of antibody proteoforms covering both the amino acid sequence and all posttranslational modifications remains a major concern. The usual mass spectrometry-based approach to achieve this goal is bottom-up proteomics, which relies on the digestion of antibodies but does not allow the diversity of proteoforms to be assessed. Middle-down and top-down approaches have recently emerged as attractive alternatives but are not yet mastered and thus used in routine by many analytical chemistry laboratories. The work described here aims at providing guidelines to achieve the best sequence coverage for the fragmentation of intact light and heavy chains generated from a simple reduction of intact antibodies using Orbitrap mass spectrometry. Three parameters were found crucial to this aim: the use of an electron-based activation technique, the multiplex selection of precursor ions of different charge states, and the combination of replicates.     

Trypsin immobilization on an ethylenediamine-based monolithic minidisk for rapid on-line peptide mass fingerprinting studies

The aim of this work was to develop a trypsin-based micro-immobilized enzyme reactor prepared on a monolithic ethylenediamine BIA Separations CIM (convective interaction media) minidisk. The micro-immobilized enzyme reactor (IMER) was integrated in a liquid chromatography system hyphenated to electrospray ionization tandem mass spectrometry to carry out on-line protein digestion and identification. The performance of this IMER was compared with that obtained using a previously developed bioreactor prepared on a conventional CIM ethylenediamine disk and with that of the commercially available Poroszyme immobilized trypsin cartridge. In this work, we showed how different proteins were identified with good recoveries using a digestion time of 10 min only.     

Comprehensive two-dimensional separation in coupling of reversed-phase chromatography with capillary isoelectric focusing followed by MALDI-MS identification using on-target digestion for intact protein analysis

A coupling of capillary RP LC as the first dimension with CIEF as the second dimension followed by MALDI-MS identification was demonstrated. Based on 2-D separation system developed by our group (Electrophoresis 2003, 24, 3289-3295), this paper focused on incorporating tryptic digestion into the top-down proteomics methodology, retaining the benefits of the top-down method. Hydrophobic layer of packing-material C18 coated with SE-30 on the MALDI-target surface was used to permit the CIEF fractions to be easily concentrated and free of ampholytes using on-target washing. Following the removal of ampholytes, on-target tryptic digestion was performed to generate PMF for protein identification. Using the proteome analytical strategy, we could obtain not only intact protein pI value but also PMF for accurate protein identification. The feasibility of the strategy was first tested with a mixture of model proteins with different pIs and molecular masses. Proteome of rat liver tissue extracts was further analyzed using the system for verification. The results have shown that the system is effective for complex proteomic analysis.     

Application of displacement chromatography for the proteome analysis of a human plasma protein fraction

It was the aim of this study to compare the performance of displacement chromatography with gradient elution chromatography both applied as the cation-exchange separation step for a proteome analysis in a bottom-up approach using multidimensional chromatography for the separation of tryptic peptides prior to their mass spectrometric analysis. The tryptic digest of the human Cohn fraction IV-4 served as a sample. For both chromatography modes commonly used operating parameters were chosen thus ensuring optimal separation results of equal sample amounts for each mode. All resulting fractions were analyzed with an HPLC-chip–LC–MS system. The eluate of the HPLC-chip column was ionized by electrospray ionization (ESI) and analyzed with an ion-trap mass spectrometer. For guaranteeing high confidence concerning the identity of the peptides, the mass spectrometric data were processed by different bioinformatic tools applying stringent criteria. By the displacement approach the total amount of identified proteins (78) was significantly higher than in the gradient mode (58). The results showed that displacement chromatography is a well suited alternative in comparison to gradient elution separation for analysis of proteomes via the bottom-up approach applying multidimensional chromatography, especially in those cases when larger quantities of proteins are available.     

Evaluation of various immobilized enzymatic microreactors coupled on-line with liquid chromatography and mass spectrometry detection for quantitative analysis of cytochrome c

An in-line procedure for protein analysis using a trypsin-based immobilized enzymatic reactor (IMER) coupled to LC-MS/MS has been developed. Various IMERs were synthesized and characterized by estimating the digestion yield of a pattern peptide in UV detection. Laboratory-made IMERs were optimized by studying the effect of different parameters as the nature of the functionalized immobilization support (silica, agarose), the amount of immobilized trypsin and the binding density. The potential of the laboratory-made IMERs were compared with a batch digestion and with a commercial trypsin-based IMER. The laboratory-made IMER based on CNBr-activated Sepharose showed the best performances in terms of digestion yields, digestion time, price and repeatability (RSD<4%). Cytochrome c was then digested on this IMER and used in-line with LC-MS. The target protein was easily recognized by the Mascot database until 17pmol injected.     

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.     

Improved in-gel approaches to generate peptide maps of integral membrane proteins with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

This paper reports studies of in-gel digestion procedures to generate MALDI-MS peptide maps of integral membrane proteins. The methods were developed for the membrane domain of the mannitol permease of E. coli. In-gel digestion of this domain with trypsin, followed by extraction of the peptides from the gel, yields only 44% sequence coverage. Since lysines and arginines are seldomly found in the membrane-spanning regions, complete tryptic cleavage will generate large hydrophobic fragments, many of which are poorly soluble and most likely contribute to the low sequence coverage. Addition of the detergent octyl-beta-glucopyranoside (OBG), at 0.1% concentration, to the extraction solvent increases the total number of peptides detected to at least 85% of the total protein sequence. OBG facilitates the recovery of hydrophobic peptides when they are SpeedVac dried during the extraction procedure. Many of the newly recovered peptides are partial cleavage products. This seems to be advantageous since it generates hydrophobic fragments with a hydrophilic solubilizing part. In-gel CNBr cleavage resulted in 5-10-fold more intense spectra, 83% sequence coverage, fully cleaved fragments and no effect of OBG. In contrast to tryptic cleavage sites, the CNBr cleavage sites are found in transmembrane segments; cleavage at these sites generates smaller hydrophobic fragments, which are more soluble and do not need OBG. With the results of both cleavages, a complete sequence coverage of the membrane domain of the mannitol permease of E. coli is obtained without the necessity of using HPLC separation. The protocols were applied to two other integral membrane proteins, which confirmed the general applicability of CNBr cleavage and the observed effects of OBG in peptide recovery after tryptic digestion.     

Specific detection of acetyl-coenzyme A by reversed-phase ion-pair high-performance liquid chromatography with an immobilized enzyme reactor

A selective chromatographic detection system for the determination of acetyl-coenzyme A (CoA) is reported. The short-chain acyl-CoA thioesters were separated by reversed-phase ion-pair high-performance liquid chromatography (HPLC), and then acetyl-CoA was selectively detected on-line with an immobilized enzyme reactor (IMER) as a post-column reactor. Thio-CoA liberated enzymatically from acetyl-CoA was determined spectrophotometrically after reaction with Ellman's reagent in the reagent stream. The IMER with phosphotransacetylase had a substrate specificity sufficient to determine acetyl-CoA and was active and stable in the mobile phase containing methanol and the ion-pair reagent. The calibration graph was linear between 0.2 and 10 nmol, with a detection limit of 0.05 nmol. This HPLC system with detection by IMER allows the selective identification and determination of acetyl-CoA in a mixture of acetoacetyl-CoA and 3-hydroxy-3-methylglutaryl-CoA, which are difficult to separate with ion-pair HPLC.     

Rapid fabrication of glass/PDMS hybrid µIMER for high throughput membrane proteomics

Mass spectrometry (MS) based proteomics has brought a radical approach to systems biology, offering a platform to study complex biological functions. However, key proteomic technical challenges remain, mainly the inability to characterise the complete proteome of a cell due to the thousands of diverse, complex proteins expressed at an extremely wide concentration range. Currently, high throughput and efficient techniques to unambiguously identify and quantify proteins on a proteome-wide scale are in demand. Miniaturised analytical systems placed upstream of MS help us to attain these goals. One time-consuming step in traditional techniques is the in-solution digestion of proteins (4-20 h). This also has other drawbacks, including enzyme autoproteolysis, low efficiency, and manual operation. Furthermore, the identification of α-helical membrane proteins has remained a challenge due to their high hydrophobicity and lack of trypsin cleavage targets in transmembrane helices. We demonstrate a new rapidly produced glass/PDMS micro Immobilised Enzyme Reactor (μIMER) with enzymes covalently immobilised onto polyacrylic acid plasma-modified surfaces for the purpose of rapidly (as low as 30 s) generating peptides suitable for MS analysis. This μIMER also allows, for the first time, rapid digestion of insoluble proteins. Membrane protein identification through this method was achieved after just 4 min digestion time, up to 9-fold faster than either dual-stage in-solution digestion approaches or other commonly used bacterial membrane proteomic workflows.     

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.

     

Intact protein separation by chromatographic and/or electrophoretic techniques for top-down proteomics

Mass spectrometry used in combination with a wide variety of separation methods is the principal methodology for proteomics. In bottom-up approach, proteins are cleaved with a specific proteolytic enzyme, followed by peptide separation and MS identification. In top-down approach intact proteins are introduced into the mass spectrometer. The ions generated by electrospray ionization are then subjected to gas-phase separation, fragmentation, fragment separation, and automated interpretation of mass spectrometric and chromatographic data yielding both the molecular weight of the intact protein and the protein fragmentation pattern. This approach requires high accuracy mass measurement analysers capable of separating the multi-charged isotopic cluster of proteins, such as hybrid ion trap-Fourier transform instruments (LTQ-FTICR, LTQ-Orbitrap). Front-end separation technologies tailored for proteins are of primary importance to implement top-down proteomics. This review intends to provide the state of art of protein chromatographic and electrophoretic separation methods suitable for MS coupling, and to illustrate both monodimensional and multidimensional approaches used for LC-MS top-down proteomics. In addition, some recent progresses in protein chromatography that may provide an alternative to those currently employed are also discussed.