RESUMEN
Blood serum and plasma are arguably the most commonly analyzed clinical samples, with dozens of proteins serving as validated biomarkers for various human diseases. Top-down proteomics may provide additional insights into disease etiopathogenesis since this approach focuses on protein forms, or proteoforms, originally circulating in blood, potentially providing access to information about relevant post-translational modifications, truncations, single amino acid substitutions, and many other sources of protein variation. However, the vast majority of proteomic studies on serum and plasma are carried out using peptide-centric, bottom-up approaches that cannot recapitulate the original proteoform content of samples. Clinical laboratories have been slow to adopt top-down analysis, also due to higher sample handling requirements. In this study, we describe a straightforward protocol for intact proteoform sample preparation based on the depletion of albumin and immunoglobulins, followed by simplified protein fractionation via polyacrylamide gel electrophoresis. After molecular weight-based fractionation, we supplemented the traditional liquid chromatography-tandem mass spectrometry (LC-MS2) data acquisition with high-field asymmetric waveform ion mobility spectrometry (FAIMS) to further simplify serum proteoform mixtures. This LC-FAIMS-MS2 method led to the identification of over 1000 serum proteoforms < 30 kDa, outperforming traditional LC-MS2 data acquisition and more than doubling the number of proteoforms identified in previous studies.
Asunto(s)
Espectrometría de Movilidad Iónica , Suero , Humanos , Espectrometría de Movilidad Iónica/métodos , Suero/química , Proteómica/métodos , Proteínas/análisis , Espectrometría de Masas/métodosRESUMEN
The high-throughput quantification of intact proteoforms using a label-free approach is typically performed on proteins in the 0-30 kDa mass range extracted from whole cell or tissue lysates. Unfortunately, even when high-resolution separation of proteoforms is achieved by either high-performance liquid chromatography or capillary electrophoresis, the number of proteoforms that can be identified and quantified is inevitably limited by the inherent sample complexity. Here, we benchmark label-free quantification of proteoforms of Escherichia coli by applying gas-phase fractionation (GPF) via field asymmetric ion mobility spectrometry (FAIMS). Recent advances in Orbitrap instrumentation have enabled the acquisition of high-quality intact and fragmentation mass spectra without the need for averaging time-domain transients prior to Fourier transform. The resulting speed improvements allowed for the application of multiple FAIMS compensation voltages in the same liquid chromatography-tandem mass spectrometry experiment without increasing the overall data acquisition cycle. As a result, the application of FAIMS to label-free quantification based on intact mass spectra substantially increases the number of both identified and quantified proteoforms without penalizing quantification accuracy in comparison to traditional label-free experiments that do not adopt GPF.
Asunto(s)
Espectrometría de Movilidad Iónica , Espectrometría de Masas en Tándem , Espectrometría de Masas en Tándem/métodos , Proteómica/métodos , Proteínas/análisis , Cromatografía Liquida , Escherichia coli/químicaRESUMEN
Field asymmetric ion mobility spectrometry (FAIMS), when used in proteomics studies, provides superior selectivity and enables more proteins to be identified by providing additional gas-phase separation. Here, we tested the performance of cylindrical FAIMS for the identification and characterization of proteoforms by top-down mass spectrometry of heterogeneous protein mixtures. Combining FAIMS with chromatographic separation resulted in a 62% increase in protein identifications, an 8% increase in proteoform identifications, and an improvement in proteoform identification compared to samples analyzed without FAIMS. In addition, utilization of FAIMS resulted in the identification of proteins encoded by lower-abundance mRNA transcripts. These improvements were attributable, in part, to improved signal-to-noise for proteoforms with similar retention times. Additionally, our results show that the optimal compensation voltage of any given proteoform was correlated with the molecular weight of the analyte. Collectively these results suggest that the addition of FAIMS can enhance top-down proteomics in both discovery and targeted applications.
Asunto(s)
Espectrometría de Movilidad Iónica , Proteómica , Espectrometría de Masas , ProteínasRESUMEN
The benefits of high field asymmetric waveform ion mobility spectrometry (FAIMS) for mass spectrometry imaging of intact proteins in thin tissue sections have been demonstrated previously. In those works, a planar FAIMS device coupled with a Thermo Elite mass spectrometer was employed. Here, we have evaluated a newly introduced cylindrical FAIMS device (the FAIMS Pro) coupled with a Thermo Fusion Lumos mass spectrometer for liquid extraction surface analysis mass spectrometry imaging of intact proteins in thin tissue sections from rat testes, kidney, and brain. The method makes use of multiple FAIMS compensation values at each location (pixel) of the imaging array. A total of 975 nonredundant protein species were detected in the testes imaging dataset, 981 in the kidney dataset, and 249 in the brain dataset. These numbers represent a 7-fold (brain) and over 10-fold (testes, kidney) improvement on the numbers of proteins previously detected in LESA FAIMS imaging, and a 10-fold to over 20-fold improvement on the numbers detected without FAIMS on this higher performance mass spectrometer, approaching the same order of magnitude as those obtained in top-down proteomics of cell lines. Nevertheless, high throughput identification within the LESA FAIMS imaging workflow remains a challenge.
Asunto(s)
Proteínas/análisis , Animales , Encéfalo , Línea Celular , Espectrometría de Movilidad Iónica , Riñón/química , Masculino , Espectrometría de Masas , Proteómica , Ratas , Ratas Wistar , Testículo/químicaRESUMEN
Multiplexed, isobaric tagging methods are powerful techniques to increase throughput, precision, and accuracy in quantitative proteomics. The dynamic range and accuracy of quantitation, however, can be limited by coisolation of tag-containing peptides that release reporter ions and conflate quantitative measurements across precursors. Methods to alleviate these effects often lead to the loss of protein and peptide identifications through online or offline filtering of interference containing spectra. To alleviate this effect, high-Field Asymmetric-waveform Ion Mobility Spectroscopy (FAIMS) has been proposed as a method to reduce precursor coisolation and improve the accuracy and dynamic range of multiplex quantitation. Here we tested the use of FAIMS to improve quantitative accuracy using previously established TMT-based interference standards (triple-knockout [TKO] and Human-Yeast Proteomics Resource [HYPER]). We observed that FAIMS robustly improved the quantitative accuracy of both high-resolution MS2 (HRMS2) and synchronous precursor selection MS3 (SPS-MS3)-based methods without sacrificing protein identifications. We further optimized and characterized the main factors that enable robust use of FAIMS for multiplexed quantitation. We highlight these factors and provide method recommendations to take advantage of FAIMS technology to improve isobaric-tag-quantification moving forward.
Asunto(s)
Espectrometría de Masas/métodos , Proteínas de Neoplasias/metabolismo , Péptidos/análisis , Proteoma/análisis , Proteómica/métodos , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Células HCT116 , Humanos , Péptidos/metabolismo , Proteoma/metabolismoRESUMEN
Liquid chromatography (LC) prefractionation is often implemented to increase proteomic coverage; however, while effective, this approach is laborious, requires considerable sample amount, and can be cumbersome. We describe how interfacing a recently described high-field asymmetric waveform ion mobility spectrometry (FAIMS) device between a nanoelectrospray ionization (nanoESI) emitter and an Orbitrap hybrid mass spectrometer (MS) enables the collection of single-shot proteomic data with comparable depth to that of conventional two-dimensional LC approaches. This next generation FAIMS device incorporates improved ion sampling at the ESI-FAIMS interface, increased electric field strength, and a helium-free ion transport gas. With fast internal compensation voltage (CV) stepping (25 ms/transition), multiple unique gas-phase fractions may be analyzed simultaneously over the course of an MS analysis. We have comprehensively demonstrated how this device performs for bottom-up proteomics experiments as well as characterized the effects of peptide charge state, mass loading, analysis time, and additional variables. We also offer recommendations for the number of CVs and which CVs to use for different lengths of experiments. Internal CV stepping experiments increase protein identifications from a single-shot experiment to >8000, from over 100 000 peptide identifications in as little as 5 h. In single-shot 4 h label-free quantitation (LFQ) experiments of a human cell line, we quantified 7818 proteins with FAIMS using intra-analysis CV switching compared to 6809 without FAIMS. Single-shot FAIMS results also compare favorably with LC fractionation experiments. A 6 h single-shot FAIMS experiment generates 8007 protein identifications, while four fractions analyzed for 1.5 h each produce 7776 protein identifications.
Asunto(s)
Espectrometría de Movilidad Iónica/instrumentación , Péptidos/análisis , Proteínas/análisis , Proteómica/instrumentación , Espectrometría de Masa por Ionización de Electrospray/instrumentación , Línea Celular , HumanosRESUMEN
Gestagens, a class of veterinary drugs also called progestogens, are synthetic hormones used to increase feed efficiency and rate of gain in heifers. The Canadian Food Inspection Agency analyzes progestogens melengestrol acetate (MGA), megestrol acetate, and chlormadinone acetate using liquid chromatography-mass spectrometry (LC-MS). Our conventional gestagen method for kidney fat has many time-consuming steps, including solid-phase extraction. A sample preparation procedure having fewer clean-up steps was developed for routine diagnostic analysis of kidney fat and provided similar results faster, and at lower cost. A confirmatory liver method for gestagens, developed using salt-assisted extraction, employed minimal clean-up steps that resulted in high chemical background at the desired lower limit of quantification (LLOQ). Differential ion mobility spectrometry, specifically high-field asymmetric waveform ion mobility spectrometry (FAIMS), was used to filter chemical background in the gas phase. The effect of the ionization probe position on FAIMS parameters, including sensitivity, is described. With LC-FAIMS-MS, chemical background for each gestagen was virtually eliminated, resulting in a quantitative liver method having the desired 0.6 ng/g LLOQ and estimated limits of detection (LODs) up to 140 times lower than LC-MS. Incurred MGA samples, analyzed using kidney fat and liver methods from the same animal, show levels within the quantitative ranges of both methods.
Asunto(s)
Acetato de Melengestrol , Progestinas , Animales , Bovinos , Femenino , Progestinas/análisis , Canadá , Cromatografía Liquida/métodos , Espectrometría de Masas/métodos , Acetato de Melengestrol/análisis , Hígado/químicaRESUMEN
Thyreostatic drugs (thyreostats) interfere with thyroid function and have been used illegally in animals slaughtered for food. Thyreostat use leads to poorer quality meat, and the drug residues can cause adverse effects in humans. These drugs, with the exception of thiouracil, do not occur naturally and require sensitive methodologies for their detection in animal tissues. Because thyreostats are low-molecular-weight polar analytes, liquid chromatography-mass spectrometry (LC-MS) is typically used for detection and, in particular, triple quadrupole mass spectrometry with selective reaction monitoring (i.e., LC-SRM). However, LC-SRM thyreostat methods suffer from chemical background noise and endogenous interferences arising from the complex tissue matrix. An improved high-field asymmetric waveform ion mobility spectrometry interface (FAIMS Pro), which separates ions based on differential ion mobility, was combined with LC-SRM to minimize these interferences. Using the same samples and conditions, LC-FAIMS-SRM showed improvements in the signal-to-noise ratio (S/N) of up to 50 times compared with our validated LC-SRM method. In addition, wider linear ranges, including substantial improvements in the lower limit of quantification (approximately an order of magnitude for tapazole and methylthiouracil), were observed with LC-FAIMS-SRM.
Asunto(s)
Residuos de Medicamentos , Espectrometría de Movilidad Iónica , Animales , Cromatografía Liquida , Espectrometría de Movilidad Iónica/métodos , Iones/química , Espectrometría de Masas en Tándem/métodosRESUMEN
Simulations show that significant ion losses occur within the commercial electrospray ionization-field asymmetric waveform ion mobility spectrometer (ESI-FAIMS) interface owing to an angular desolvation gas flow and because of the impact of the FAIMS carrier gas onto the inner rf (radio frequency) electrode. The angular desolvation gas flow diverts ions away from the entrance plate orifice while the carrier gas annihilates ions onto the inner rf electrode. A novel ESI-FAIMS interface is described that optimizes FAIMS gas flows resulting in large improvements in transmission. Simulations with the bromochloroacetate anion showed an improvement of ~9-fold to give ~70% overall transmission). Comparable transmission improvements were attained experimentally for six peptides (2+) in the range of m/z 404.2 to 653.4 at a chromatographic flow rate of 300 nL/min. Selected ion chromatograms (SIC) from nano-LC-FAIMS-MS analyses showed 71% (HLVDEPQNLIK, m/z 653.4, 2+) to 95% (LVNELTEFAK, m/z 582.3, 2+) of ion signal compared with ion signal in the SIC from LC-MS analysis. IGSEVYHNLK (580.3, 2+) showed 24% more ion signal compared with LC-MS and is explained by enhanced desolvation in FAIMS. A 3-10 times lower limits of quantitation (LOQ) (<15% RSD) was achieved for chemical noise limited peaks with FAIMS. Peaks limited by ion statistics showed subtle improvement in RSD and yielded comparable LOQ to that attained with nano-LC-MS (without FAIMS). These improvements were obtained using a reduced FAIMS separation gap (from 2.5 to 1.5 mm) that results in a shorter residence time (13.2 ms ± 3.9 ms) and enables the use of a helium free transport gas (100% nitrogen).