Analysis Of Intact Proteins Research Articles

PEPPI-MS: gel-based sample pre-fractionation for deep top-down and middle-down proteomics.

Top-down analysis of intact proteins and middle-down analysis of proteins subjected to limited digestion require efficient detection of traces of proteoforms in samples, necessitating the reduction of sample complexity by thorough pre-fractionation of the proteome components in the sample. SDS-PAGE is a simple and inexpensive high-resolution protein-separation technique widely used in biochemical and molecular biology experiments. Although its effectiveness for sample preparation in bottom-up proteomics has been proven, establishing a method for highly efficient recovery of intact proteins from the gel matrix has long been a challenge for its implementation in top-down and middle-down proteomics. As a much-awaited solution to this problem, we present an experimental protocol for efficient proteoform fractionation from complex biological samples using passively eluting proteins from polyacrylamide gels as intact species for mass spectrometry (PEPPI-MS), a rapid method for extraction of intact proteins separated by SDS-PAGE. PEPPI-MS allows recovery of proteins below 100 kDa separated by SDS-PAGE in solution with a median efficiency of 68% within 10 min and, unlike conventional electroelution methods, requires no special equipment, contributing to a remarkably economical implementation. The entire protocol from electrophoresis to protein purification can be performed in <5 h. By combining the resulting PEPPI fraction with other protein-separation techniques, such as reversed-phase liquid chromatography and ion mobility techniques, multidimensional proteome separations for in-depth proteoform analysis can be easily achieved.

Capillary Isoelectric Focusing of Proteins and Peptides Using an In-Line cIEF-ESI Interface with Improved MS Characteristics.

Intact protein analysis using mass spectrometry (MS) is an important technique to characterize and provide a comprehensive overview of protein complexity. It is also the basis of "top-down" approaches in proteomics to describe the proteoforms of single protein's post-translational modifications (PTMs). MS-based analysis of intact proteins benefits from high-resolution separations prior to electrospray ionization. Capillary isoelectric focusing (cIEF) is a high-resolution separation for proteins and peptides which is capable of separating proteoforms. MS detection coupled to cIEF can separate, detect, and characterize proteoforms at the molecular level. However, cIEF with MS detection is a compromised process. The concentration of ampholytes required for cIEF is mutually exclusive with mass spectrometer contamination. We have improved an online cIEF-ESI-MS interface to reduce (desalt) amino acid ampholytes in-line after cIEF and prior to electrospray ionization. In proof of principle experiments, >90% increase in area under the curve of the electropherograms was observed with the interface compared to without the interface. Protein standards including proteoforms of cytochrome C, myoglobin, and α-casein were separated and resolved with high reproducibility. The interface did not compromise the linearity of the cIEF pH gradient separations, achieving a high linearity with a R2 of 0.99. In addition, a tryptic digest of BSA demonstrates baseline resolution of peptides with as little as 0.02 pI unit difference and a full width at half-maximum average of 7.1 s.

Top-down proteomics of venoms from five Micrurus species from Costa Rica: Comparative composition of phospholipase A2-rich vs three-finger toxin-rich phenotypes

Coralsnakes of the genus Micrurus include more than 80 species distributed in the American continent. They produce potent neurotoxic venoms acting at the neuromuscular junction and potentially leading to respiratory paralysis and death. The vast majority of proteins in coralsnake venoms belong to the three-finger toxin (3FTx) and the group I phospholipase A2 (PLA2) families. Previous studies using ‘bottom-up’ proteomic strategies have revealed a compositional dichotomy of toxin expression by which different Micrurus species display a predominance of either 3FTx or PLA2 proteins in their venoms, possibly linked to the phylogeographic structure of the genus radiation. ‘Top-down’ proteomics (TDP) allows the direct analysis of intact proteins in a high resolution mass spectrometer, circumventing the limitations of the ‘peptide-to-protein inference problem’ inherent to the bottom-up approach. Here, we analyzed the venoms of five out of the six Micrurus species that inhabit Costa Rica, by using a TDP approach. Results unveil venom proteoforms that are shared between these species, and provide additional insights into the variable compositional complexity of these venoms and relationships to their 3FTx/PLA2 dichotomy.

Mass Spectrometry–Based Proteomics for the Masses: Peptide and Protein Identification in the Hunt Laboratory During the 2000’s

There has been a rapid increase in the number of individuals utilizing mass spectrometry-based proteomics to study complex biological systems and questions since the start of the 2000's. Building off the advancements in ionization and liquid chromatography scientists continued to push towards technology that would enable in-depth analysis of biological specimen. Donald F Hunt and the Hunt laboratory were major contributors to this effort with their work on improving upon existing Fourier Transform MS, development of electron transfer dissociation, and continued work on ion-ion reactions to improve intact protein analysis. Collaboration with other instrumentation laboratories and instrument companies led to the sharing of technology and eventual commercialization providing greater access. Additionally, the Hunt laboratory spread the gospel of MS-based proteomics through collaborations that lasted decades with other scientists who were experts in immunology, cellular signaling, epigenetics, and other fascinating fields. This article attempts to highlight the many contributions of Don and the Hunt laboratory to peptide and protein identification since the year 2000.

Qualitative and quantitative characterization of elastin-like polypeptides combining low-PH/low-temperature bottom-up and intact protein liquid chromatography mass spectrometric analysis.

Elastin-like polypeptides (ELPs) are elastic and thermoresponsive biopolymers composed of VPGXG repeats (X can be any amino acid except proline), used in biomedical applications, for example, tissue engineering and drug delivery. As different variants of ELP are mostly produced fermentatively, there is a need for the development of analysis methods that allow for absolute protein quantification in both complex matrices and purified samples and MW determination of the final products. ELPs were intracellularly expressed in Escherichia coli quantified after cell lysis and enzymatic digestion using a proline-specific protease ProAlanase (Promega) at acidic conditions. Resulting peptides were separated by liquid chromatography, and mass spectrometry analysis was conducted by electrospray ionization high-resolution mass spectrometry using an Orbitrap mass spectrometer. The addition of a stable isotopically labeled internal standard enabled quantification in complex matrices. Prior to intact mass analysis, ELPs were purified from fermentation broth by inverse temperature cycling. Intact protein analysis was performed using reversed-phase liquid chromatography, and mass spectrometry analysis was conducted by electrospray ionization high-resolution mass spectrometry using a time-of-flight mass spectrometer. Absolute quantification of ELPs was achieved by utilizing ELP-specific properties, that is, proline-rich, soluble at low pH and low temperature. The repetitive nature of ELPs allows for sensitivity increase and use of higher dilution factors to minimize the matrix effects. Despite the lack of amino acids with charged side chains (Arg, His, Lys, Asp, and Glu) in ELP, we demonstrated successful intact protein analysis using reversed-phase LC coupled to electrospray ionization TOF MS. Moreover, truncated protein forms could be chromatographically separated and characterized as well as N-terminal modifications. Both methods combined enabled quantitative and qualitative characterization of fermentatively produced ELPs.

Immunoaffinity Intact-Mass Spectrometry for the Detection of Endogenous Concentrations of the Acetylated Protein Tumor Biomarker Neuron Specific Enolase.

Intact-mass spectrometry has huge potential for clinical application, as it enables both quantitative and qualitative analysis of intact proteins and possibly unlocks additional pathophysiological information via, e.g., detection of specific post-translational modifications (PTMs). Such valuable and clinically useful selectivity is typically lost during conventional bottom-up mass spectrometry. We demonstrate an innovative immunoprecipitation protein enrichment assay coupled to ultrahigh performance liquid chromatography quadrupole time-of-flight high resolution mass spectrometry (UPLC-QToF-HRMS) for the fast and simple identification of the protein tumor marker Neuron Specific Enolase Gamma (NSEγ) at low endogenous concentrations in human serum. Additionally, using the combination of immunoaffinity purification with intact mass spectrometry, the presence of NSEγ in an acetylated form in human serum was detected. This highlights the unique potential of immunoaffinity intact mass spectrometry in clinical diagnostics.

Top-Down Proteomics Analysis of Picogram-Level Complex Samples Using Spray-Capillary-Based Capillary Electrophoresis-Mass Spectrometry.

Proteomics analysis of mass-limited samples has become increasingly important for understanding biological systems in physiologically relevant contexts such as patient samples, multicellular organoids, spheroids, and single cells. However, relatively low sensitivity in top-down proteomics methods makes their application to mass-limited samples challenging. Capillary electrophoresis (CE) has emerged as an ideal separation method for mass-limited samples due to its high separation resolution, ultralow detection limit, and minimal sample volume requirements. Recently, we developed "spray-capillary", an electrospray ionization (ESI)-assisted device, that is capable of quantitative ultralow-volume sampling (e.g., pL-nL level). Here, we developed a spray-capillary-CE-MS platform for ultrasensitive top-down proteomics analysis of intact proteins in mass-limited complex biological samples. Specifically, to improve the sensitivity of the spray-capillary platform, we incorporated a polyethylenimine (PEI)-coated capillary and optimized the spray-capillary inner diameter. Under optimized conditions, we successfully detected over 200 proteoforms from 50 pg of E. coli lysate. To our knowledge, the spray-capillary CE-MS platform developed here represents one of the most sensitive detection methods for top-down proteomics. Furthermore, in a proof-of-principle experiment, we detected 261 ± 65 and 174 ± 45 intact proteoforms from fewer than 50 HeLa and OVCAR-8 cells, respectively, by coupling nanodroplet-based sample preparation with our optimized CE-MS platform. Overall, our results demonstrate the capability of the modified spray-capillary CE-MS platform to perform top-down proteomics analysis on picogram amounts of samples. This advancement presents the possibility of meaningful top-down proteomics analysis of mass-limited samples down to the level of single mammalian cells.

Analysis of intact venom proteins with capillary zone electrophoresis - mass spectrometry

In this work we demonstrated the favourable analytical performance of capillary zone electrophoresis coupled with mass spectrometry (CZE-MS) for the intact protein analysis of similar venom samples. Venom provides a characteristic feature (fingerprint) of a given snake. Using 1 M formic acid (pH = 1.9) as background electrolyte, minimal adsorption and narrow peaks shapes - thus good separation efficiencies - were obtained for the protein components of the venom samples. The precision of migration times and peak areas were 1.9–2.8 RSD% and 0.8–7.2 RSD%, respectively and the theoretical plate numbers were 32000–238000 for peaks having signal-to-noise ratio (S/N) larger than 50.More than 250 different neuropeptides (7–10 kDa) were detected in the venoms obtained from snakes of 9 different subspecies (belonging either to Naja or Dendroaspis species). The protein contents of the venoms of the same subspecies collected from different geographical regions are similar and differ only in a few (less than 10 %) components. However, the venoms collected from different subspecies (but from the same species) exhibit very different protein patterns: no matches were found in the molecular weights of proteins.

Toward Unrivaled Chromatographic Resolving Power in Proteomics: Design and Development of Comprehensive Spatial Three-Dimensional Liquid-Phase Separation Technology.

Spatial comprehensive three-dimensional chromatography (3D-LC) offers an innovative approach to achieve unprecedented resolving power in terms of peak capacity and sample throughput. This advanced technique separates components within a 3D separation space, where orthogonal retention mechanisms are incorporated. The parallel development of the second- and third-dimension stages effectively overcomes the inherent limitation of conventional multidimensional approaches, where sampled fractions are analyzed sequentially. This review focuses on the design aspects of the microchip for spatial 3D-LC and the selection of orthogonal separation modes to enable the analysis of intact proteins. The design considerations for the flow distributor and channel layout are discussed, along with various approaches to confine the flow during the subsequent development stages. Additionally, the integration of stationary phases into the microchip is addressed, and interfacing to mass spectrometry detection is discussed. According to Pareto optimality, the integration of isoelectric focusing, size-exclusion chromatography, and reversed-phase chromatography in a spatial 3D-LC approach is predicted to achieve an exceptional peak capacity of over 30,000 within a 1-h analysis, setting a new benchmark in chromatographic performance.

AuNPs/CNC Nanocomposite with A "Dual Dispersion" Effect for LDI-TOF MS Analysis of Intact Proteins in NSCLC Serum Exosomes.

Detecting exosomal markers using laser desorption/ionization time-of-flight mass spectrometry (LDI-TOF MS) is a novel approach for examining liquid biopsies of non-small cell lung cancer (NSCLC) samples. However, LDI-TOF MS is limited by low sensitivity and poor reproducibility when analyzing intact proteins directly. In this report, gold nanoparticles/cellulose nanocrystals (AuNPs/CNC) is introduced as the matrix for direct analysis of intact proteins in NSCLC serum exosomes. AuNPs/CNC with "dual dispersion" effects dispersed and stabilized AuNPs and improved ion inhibition effects caused by protein aggregation. These features increased the signal-to-noise ratio of [M+H]+ peaks by two orders of magnitude and lowered the detection limit of intact proteins to 0.01mgmL-1. The coefficient of variation with or without AuNPs/CNC is measured as 10.2% and 32.5%, respectively. The excellent reproducibility yielded a linear relationship (y=15.41x - 7.983, R2=0.989) over the protein concentration range of 0.01 to 20mgmL-1. Finally, AuNPs/CNC-assisted LDI-TOF MS provides clinically relevant fingerprint information of exosomal proteins in NSCLC serum, and characteristic proteins S100 calcium-binding protein A10, Urokinase plasminogen activator surface receptor, Plasma protease C1 inhibitor, Tyrosine-protein kinase Fgr and Mannose-binding lectin associated serine protease 2 represented excellent predictive biomarkers of NSCLC risk.

Enhancing Top-Down Analysis of Proteins by Combining Ultraviolet Photodissociation (UVPD), Proton-Transfer Charge Reduction (PTCR), and Gas-Phase Fractionation to Alleviate the Impact of Nondissociated Precursor Ions.

Recent advances in top-down mass spectrometry strategies continue to improve the analysis of intact proteins. 193 nm ultraviolet photodissociation (UVPD) is one method well-suited for top-down analysis. UVPD is often performed using relatively low photon flux in order to limit multiple-generation dissociation of fragment ions and maximize sequence coverage. Consequently, a large portion of the precursor ion survives the UVPD process, dominates the spectrum, and may impede identification of fragment ions. Here, we explore the isolation of subpopulations of fragment ions lower and higher than the precursor ion after UVPD as a means to eliminate the impact of the surviving precursor ion on the detection of low abundance fragment ions. This gas-phase fractionation method improved sequence coverage harvested from fragment ions found in the m/z regions lower and higher than the precursor by an average factor of 1.3 and 2.3, respectively. Combining this gas-phase fractionation method with proton transfer charge reduction (PTCR) further increased the sequence coverage obtained from these m/z regions by another factor of 1.3 and 1.4, respectively. Implementing a post-UVPD fractionation + PTCR strategy with six fractionation events resulted in a sequence coverage of 75% for enolase, the highest reported for 193 nm UVPD.

The need for open and FAIR data in top-down proteomics.

In recent years, there has been a tremendous evolution in the high-throughput, tandem mass spectrometry-based analysis of intact proteins, also known as top-down proteomics (TDP). Both hardware and software have developed to the point that the technique has largely entered the mainstream, and large-scale, ambitious, multi-laboratory initiatives have started to make their appearance in the literature. For this, however, more convenient and robust data sharing and reuse will be required. Walzer etal. have created TopDownApp, a customisable, open platform for visualisation and analysis of TDP data, which they hope will be a step in this direction. As they point out, other benefits of such data sharing and interoperability would include reanalysis of published datasets, as well as the prospect of using large amounts of data to train machine learning algorithms. In time, this work could prove to be a valuable resource in the move towards a future of greater TDP data findability, accessibility, interoperability and reusability.

User-friendly platform for analysis of high mass intact proteins and glycopeptides by laser desorption/ionization-mass spectrometry based on copper oxide particles

Matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) based on micro/nanostructured materials with different natures has received increasing attention for the analysis of a wide variety of analytes. However, up to now, only a few studies have shown the application of simple platforms in MALDI-MS for the identification of intact proteins. The present work reports on the application of copper oxide particles (Cu2O PS), obtained by a greener route, in combination with low amounts of 2,5-dihydroxybenzoic acid (DHB) as a novel hybrid platform. The combined Cu2O PS@DHB matrix, containing only 2.5 mg mL−1 of particles and 10 mg mL−1 of DHB, was easily applicable in MALDI-MS without surface modification of target plates. Under optimal conditions, the analysis of intact proteins up to 150,000 Da was possible, including immunoglobulin G, bovine serum albumin, and cytochrome C with adequate spot-to-spot signal reproducibility (RSD < 10%). In addition, the analysis of glycopeptides from IgG digests was carried out to prove the multipurpose application of the Cu2O PS@DHB platform in the low m/z range (2500–3000 Da). From the obtained results, it can be concluded that the optical and surface properties of as-synthesized Cu2O PS are likely to be responsible for the superior performance of Cu2O PS@DHB in comparison with conventional matrices. In this sense, the proposed user-friendly methodology opens up the prospect for possible implementation in bioanalysis and diagnostic research.Graphical

TopDownApp: An open and modular platform for analysis and visualisation of top-down proteomics data.

Although Top-down (TD) proteomics techniques, aimed at the analysis of intact proteins and proteoforms, are becoming increasingly popular, efforts are needed at different levels to generalise their adoption. In this context, there are numerous improvements that are possible in the area of open science practices, including a greater application of the FAIR (Findable, Accessible, Interoperable, and Reusable) data principles. These include, for example, increased data sharing practices and readily available open data standards. Additionally, the field would benefit from the development of open data analysis workflows that can enable data reuse of public datasets, something that is increasingly common in other proteomics fields.

Advancing Intact Protein Quantitation with Updated Deconvolution Routines.

Analysis of intact proteins by mass spectrometry enables direct quantitation of the specific proteoforms present in a sample and is an increasingly important tool for biopharmaceutical and academic research. Interpreting and quantifying intact protein species from mass spectra typically involves many challenges including mass deconvolution and peak processing as well as determining optimal spectral averaging parameters and matching masses to theoretical proteoforms. Each of these steps can present informatic hurdles, as parameters often need to be tailored specifically to the data sets. To reduce intact mass deconvolution data analysis burdens, we built upon the widely used "sliding window" mass deconvolution technique with several additional concepts. First, we found that how spectra are averaged and the overlap in spectral windows can be tuned to favor either sensitivity or speed. A multiple window averaging approach was found to be the most effective way to increase mass detection and yielded a >2-fold increase in the number of masses detected. We also developed a targeted feature-finding routine that boosted sensitivity by >2-fold, decreased coefficient of variation across replicates by 50%, and increased the quality of mass elution profiles through 3-fold more detected time points. Lastly, we furthered existing approaches for annotating detected masses with potential proteoforms through spectral fitting for possible proteoform family modifications and network viewing. These proteoform annotation approaches ultimately produced a more accurate way of finding related, but previously unknown proteoforms from intact mass-only data. Together, these quantitation workflow improvements advance the information obtainable from intact protein mass spectrometry analyses.

Exploiting ion-mobility mass spectrometry for unraveling proteome complexity.

Ion mobility spectrometry-mass spectrometry (IMS-MS) is experiencing rapid growth in proteomic studies, driven by its enhancements in dynamic range and throughput, increasing the quantitation precision, and the depth of proteome coverage. The core principle of ion mobility spectrometry is to separate ions in an inert gas under the influence of an electric field based on differences in drift time. This minireview provides an introduction to IMS operation modes and a description of advantages and limitations is presented. Moreover, the principles of trapped IMS-MS (TIMS-MS), including parallel accumulation-serial fragmentation are discussed. Finally, emerging applications linked to TIMS focusing on sample throughput (in clinical proteomics) and sensitivity (single-cell proteomics) are reviewed, and the possibilities of intact protein analysis are discussed.

Capillary electrophoresis-mass spectrometry for intact protein analysis: Pharmaceutical and biomedical applications (2018-March 2023).

Capillary electrophoresis is recognized as a valued separation technique for its high separation efficiency, low sample consumption, good economic and ecological aspects, reproducibility, and complementarity to traditional liquid chromatography techniques. Capillary electrophoresis experiments are generally performed utilizing optical detection, such as ultraviolet or fluorescence detectors. However, in order to provide structural information, capillary electrophoresis hyphenated to highly sensitive and selective mass spectrometry has been developed to overcome the limitations of optical detections. Capillary electrophoresis-mass spectrometry is increasingly popular in protein analysis, including biopharmaceutical and biomedical research. It is frequently applied for the determination of physicochemical and biochemical parameters of proteins, offers excellent performance for in-depth characterizations of biopharmaceuticals at various levels of analysis, and has been also already proven as a promising tool in biomarker discovery. In this review, we focus on the possibilities and limitations of capillary electrophoresis-mass spectrometry for protein analysis at their intact level. Various capillary electrophoresis modes and capillary electrophoresis-mass spectrometry interfaces, as well as approaches to prevent protein adsorption and to enhance sample loading capacity, are discussed and the recent (2018-March 2023) developments and applications in the field of biopharmaceutical and biomedical analysis are summarized.

Ultra-High-Throughput and Low-Volume Analysis of Intact Proteins with LAP-MALDI MS.

High-throughput (HTP) mass spectrometry (MS) is a rapidly growing field, with many techniques evolving to accommodate ever increasing sample analysis rates. Many of these techniques, such as AEMS and IR-MALDESI MS, require volumes of at least 20-50 μL for analysis. Here, liquid atmospheric pressure-matrix-assisted laser desorption/ionization (LAP-MALDI) MS is presented as an alternative for ultra-high-throughput analysis of proteins requiring only femtomole quantities of protein in 0.5 μL droplets. By moving a 384-well microtiter sample plate with a high-speed XY-stage actuator, sample acquisition rates of up to 10 samples per second have been achieved at a data acquisition rate of 200 spectra per scan. It is shown that protein mixture solutions with concentrations of ≤2 μM can be analyzed at this speed, while individual protein solutions can be analyzed at concentrations of ≤0.2 μM. Thus, LAP-MALDI MS provides a promising platform for multiplexed HTP protein analysis.

Direct deamidation analysis of intact adeno-associated virus serotype 9 capsid proteins using reversed-phase liquid chromatography

Recombinant adeno-associated viral (AAV) vectors have taken center stage as gene delivery vehicles for gene therapy. Asparagine deamidation of AAV capsid proteins has been reported to reduce vector stability and potency of AAV gene therapy products. Deamidation of asparagine residue is a common post-translational modification of proteins that is detected and quantified by liquid chromatography-tandem mass spectrometry (LC-MS)-based peptide mapping. However, artificial deamidation can be spontaneously induced during sample preparation for peptide mapping prior to LC-MS analysis. We have developed an optimized sample preparation method to reduce and minimize deamidation artifacts induced during sample preparation for peptide mapping, which typically takes several hours to complete. To shorten turnaround time of deamidation results and to avoid artificial deamidation, we developed orthogonal RPLC-MS and RPLC-fluorescence detection methods for direct deamidation analysis at the intact AAV9 capsid protein level to routinely support downstream purification, formulation development, and stability testing. Similar trends of increasing deamidation of AAV9 capsid proteins in stability samples were observed at the intact protein level and peptide level, indicating that the developed direct deamidation analysis of intact AAV9 capsid proteins is comparable to the peptide mapping-based deamidation analysis and both methods are suitable for deamidation monitoring of AAV9 capsid proteins.

Software-Assisted Data Processing Workflow for Intact Glycoprotein Mass Spectrometry.

Intact protein analysis by mass spectrometry is important for several applications such as assessing post-translational modifications and biotransformation. In particular, intact protein analysis allows the detection of proteoforms that are commonly missed by other approaches such as proteolytic digestion followed by bottom-up analysis. Two quantification methods are mainly used for intact protein data quantification, namely the extracted ion and deconvolution approaches. However, a consensus with regard to a single best practice for intact protein data processing is lacking. Furthermore, many data processing tools are not fit-for-purpose and, as a result, the analysis of intact proteins is laborious and lacks the throughput required to be implemented for the analysis of clinical cohorts. Therefore, in this study, we investigated the application of a software-assisted data analysis and processing workflow in order to streamline intact protein integration, annotation, and quantification via deconvolution. In addition, the assessment of orthogonal data sets generated via middle-up and bottom-up analysis enabled the cross-validation of cleavage proteoform assignments present in seminal prostate-specific antigen (PSA). Furthermore, deconvolution quantification of PSA from patients' urine revealed results that were comparable with manually performed quantification based on extracted ion electropherograms. Overall, the presented workflow allows fast and efficient processing of intact protein data. The raw data is available on MassIVE using the identifier MSV000086699.