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.

An automated spray-capillary platform for the microsampling and CE-MS analysis of picoliter- and nanoliter-volume samples.

Capillary electrophoresis mass spectrometry (CE-MS) is an emerging analytical tool for microscale biological sample analysis that offers high separation resolution, low detection limit, and low sample consumption. We recently developed a novel microsampling device, "spray-capillary," for quantitative low-volume sample extraction (as low as 15 pL/s) and online CE-MS analysis. This platform can efficiently analyze picoliter samples (e.g., single cells) with minimal sample loss and no additional offline sample-handling steps. However, our original spray-capillary-based experiments required manual manipulation of the sample inlet for sample collection and separation, which is time consuming and requires proficiency in device handling. To optimize the performance of spray-capillary CE-MS analysis, we developed an automated platform for robust, high-throughput analysis of picoliter samples using a commercially available CE autosampler. Our results demonstrated high reproducibility among 50 continuous runs using the standard peptide angiotensin II (Ang II), with an RSD of 14.70% and 0.62% with respect to intensity and elution time, respectively. We also analyzed Ang II using varying injection times to evaluate the capability of the spray-capillary to perform quantitative sampling and found high linearity for peptide intensity with respect to injection time (R2 > 0.99). These results demonstrate the capability of the spray-capillary sampling platform for high-throughput quantitative analysis of low-volume, low-complexity samples using pressure elution (e.g., direct injection). To further evaluate and optimize the automated spray-capillary platform to analyze complex biological samples, we performed online CE-MS analysis on Escherichia coli lysate digest spiked with Ang II using varying injection times. We maintained high linearity of intensity with respect to injection time for Ang II and E. coli peptides (R2 > 0.97 in all cases). Furthermore, we observed good CE separation and high reproducibility between automated runs. Overall, we demonstrated that the automated spray-capillary CE-MS platform can efficiently and reproducibly sample picoliter and nanoliter biological samples for high-throughput proteomics analysis.

Optimization of Higher-Energy Collisional Dissociation Fragmentation Energy for Intact Protein-Level Tandem Mass Tag Labeling.

Isobaric chemical tag labeling (e.g., TMT) is a commonly used approach in quantitative proteomics, and quantification is enabled through detection of low-mass reporter ions generated after MS2 fragmentation. Recently, we have introduced and optimized an intact protein-level TMT labeling platform that demonstrated >90% labeling efficiency in complex samples with top-down proteomics. Higher-energy collisional dissociation (HCD) is commonly utilized for isobaric tag-labeled peptide fragmentation because it produces accurate reporter ion intensities and avoids loss of low mass ions. HCD energies have been optimized for isobaric tag labeled-peptides but have not been systematically evaluated for isobaric tag-labeled intact proteins. In this study, we report a systematic evaluation of normalized HCD fragmentation energies (NCEs) on TMT-labeled HeLa cell lysate using top-down proteomics. Our results suggested that reporter ions often result in higher ion intensities at higher NCEs. Optimal fragmentation of intact proteins for identification, however, required relatively lower NCE. We further demonstrated that a stepped NCE scheme with energies from 30% to 50% resulted in optimal quantification and identification of TMT-labeled HeLa proteins. These parameters resulted in an average reporter ion intensity of ∼4E4 and average proteoform spectrum matches (PrSMs) of >1000 per RPLC-MS/MS run with a 1% false discovery rate (FDR) cutoff.

Elucidating Protein-Ligand Interactions in Cell Lysates Using High-Throughput Hydrogen-Deuterium Exchange Mass Spectrometry with Integrated Protein Thermal Depletion.

Hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS) is a powerful technique for the characterization of protein-ligand interactions. Currently, there is a growing need for breakthroughs in the application of HDX-MS analysis to protein-ligand interactions in highly complex biological samples such as cell lysates. However, HDX-MS analysis in such systems suffers from extreme spectral complexity as a result of high sample complexity and limited LC separation power due to the traditional use of short LC gradients. Here, we introduced protein thermal depletion (PTD) to reduce protein complexity in E. coli cell lysate for our subzero-temperature long gradient UPLC-HDX-MS platform (PTD-HDX-MS) to facilitate high-throughput analysis of protein-ligand interactions in cell lysates. We spiked bovine carbonic anhydrase II (CaII) and its inhibitor acetazolamide (AZM) into E. coli cell lysate as a model system in our study. We demonstrated that PTD at 60 °C greatly reduces protein complexity in cell lysates, while the AZM-targeted CaII complex remains in solution due to improved thermal stability upon binding. Using both PTD to reduce sample complexity and subzero-temperature long gradient UPLC to boost LC separation power, we successfully elucidated the interaction sites between AZM and CaII in E. coli cell lysate from the high-throughput HDX-MS analysis of thousands of deuterated peptides from hundreds of proteins. Our results highlight the great promise of the PTD-HDX-MS platform for the identification of ligand targets and characterization of protein-ligand interactions in highly complex biological samples such as cell lysates.

The Deuterium Calculator: An Open-Source Tool for Hydrogen-Deuterium Exchange Mass Spectrometry Analysis.

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a powerful protein footprinting technique to study protein dynamics and binding; however, HDX-MS data analysis is often challenging and time-consuming. Moreover, the HDX community is expanding to investigate multiprotein and highly complex protein systems which further complicates data analysis. Thus, a simple, open-source software package designed to analyze large and highly complex protein systems is needed. In this vein, we have developed "The Deuterium Calculator", a Python-based software package for HDX-MS data analysis. The Deuterium Calculator is capable of differential and nondifferential HDX-MS analysis, produces standardized data files according to recommendations from the International Conference on Hydrogen-Exchange Mass Spectrometry (IC-HDX) to increase transparency in data analysis, and generates Woods' plots for statistical analysis and data visualization. This standard output can be used to perform time dependent deuteration studies and for the study of protein folding kinetics or differential uptake. Moreover, The Deuterium Calculator is capable of performing these analyses on large HDX-MS data sets (e.g., LC-HDX-MS from cell lysate digest). The Deuterium Calculator is freely available for download at https://github.com/OUWuLab/TheDeuteriumCalculator.git. Data are available via ProteomeXchange with identifier PXD036813.

Multidimensional Separations in Top-Down Proteomics.

Top-down proteomics (TDP) identifies, quantifies, and characterizes proteins at the intact proteoform level in complex biological samples to understand proteoform function and cellular mechanisms. However, analyzing complex biological samples using TDP is still challenging due to high sample complexity and wide dynamic range. High-resolution separation methods are often applied prior to mass spectrometry (MS) analysis to decrease sample complexity and increase proteomics throughput. These separation methods, however, may not be efficient enough to characterize low abundance intact proteins in complex samples. As such, multidimensional separation techniques (combination of two or more separation methods with high orthogonality) have been developed and applied that demonstrate improved separation resolution and more comprehensive identification in TDP. A suite of multidimensional separation methods that couple various types of liquid chromatography (LC), capillary electrophoresis (CE), and/or gel electrophoresis-based separation approaches have been developed and applied in TDP to analyze complex biological samples. Here, we reviewed multidimensional separation strategies employed for TDP, summarized current applications, and discussed the gaps that may be addressed in the future.

Separation methods in single-cell proteomics: RPLC or CE?

Cellular heterogeneity is commonly investigated using single-cell genomics and transcriptomics to investigate biological questions such as disease mechanism, therapeutic screening, and genomic and transcriptomic diversity between cellular populations and subpopulations at the cellular level. Single-cell mass spectrometry (MS)-based proteomics enables the high-throughput examination of protein expression at the single-cell level with wide applicability, and with spatial and temporal resolution, applicable to the study of cellular development, disease, effect of treatment, etc. The study of single-cell proteomics has lagged behind genomics and transcriptomics largely because proteins from single-cell samples cannot be amplified as DNA and RNA can using well established techniques such as PCR. Therefore, analytical methods must be robust, reproducible, and sensitive enough to detect the very small amount of protein within a single cell. To this end, nearly every step of the proteomics process has been extensively altered and improved to facilitate the proteomics analysis of single cells including cell counting and sorting, lysis, protein digestion, sample cleanup, separation, MS data acquisition, and data analysis. Here, we have reviewed recent advances in single-cell protein separation using nano reversed phase liquid chromatography (nRPLC) and capillary electrophoresis (CE) to inform application driven selection of separation techniques in the laboratory setting.

A benchmarking protocol for intact protein-level Tandem Mass Tag (TMT) labeling for quantitative top-down proteomics.

Isobaric chemical tag labeling for quantification of intact proteins in complex samples is limited due to the tendency of intact proteins precipitate under labeling conditions and increased sample complexity as a result of side products (i.e., incomplete labeling or labeling of unintended residues). To reduce precipitation under labeling conditions, we developed a technique to remove large proteoforms that allowed for the labeling and characterization of small proteoforms (<35 kDa) using top-down proteomics. We also systematically optimized protein-level Tandem Mass Tag (TMT) labeling conditions to obtain optimal labeling parameters for complex samples. Here, we present a benchmarking protocol for protein-level TMT labeling for quantitative top-down proteomics, including complex intact protein sample preparation, protein-level TMT labeling, top-down LC/MS analysis, and TMT reporter ion quantification.•An optimized protocol for protein-level TMT labeling in complex sample.•Limits production of incorrectly labeled side products for minimization of spectral complexity.•A guideline for isobaric chemical tag quantification in top-down proteomics.

Optimization of protein-level tandem mass tag (TMT) labeling conditions in complex samples with top-down proteomics.

Isobaric chemical tag labels (e.g., iTRAQ and TMT) have been extensively utilized as a standard quantification approach in bottom-up proteomics, which provides high multiplexing capacity and enables MS2-level quantification while not complicating the MS1 scans. We recently demonstrated the feasibility of intact protein TMT labeling for the identification and quantification with top-down proteomics of smaller intact proteoforms (<35 kDa) in complex biological samples through the removal of large proteins prior to labeling. Still, the production of side products during TMT labeling (i.e., incomplete labeling or labeling of unintended residues) complicated the analysis of complex protein samples. In this study, we systematically evaluated the protein-level TMT labeling reaction parameters, including TMT-to-protein mass ratio, pH/concentration of quenching buffer, protein concentration, reaction time, and reaction buffer. Our results indicated that: (1) high TMT-to-protein mass ratio (e.g., 8:1, 4:1), (2) high pH/concentration of quenching buffer (pH > 9.1, final hydroxylamine concentration >0.3%), and (3) high protein concentration (e.g., > 1.0 µg/µL) resulted in optimal labeling efficiency and minimized production of over/underlabeled side products. >90% labeling efficiency was achieved for E. coli cell lysate after optimization of protein-level TMT labeling conditions. In addition, a double labeling approach was developed for efficiently labeling limited biological samples with low concentrations. This research provides practical guidance for efficient TMT labeling of complex intact protein samples, which can be readily adopted in the high-throughput quantification top-down proteomics.

RPLC-RPLC-MS/MS for Proteoform Identification.

Top-down proteomics methods have a distinct advantage over bottom-up methods in that they analyze intact proteins rather than digested peptides which can result in loss of information regarding the intact protein. However, the analysis of intact proteins using top-down proteomics methods has been impeded by the low resolution of typical separation approaches applied in bottom-up proteomics studies. To increase the coverage of intact proteomes, orthogonal, two-dimensional separation techniques have been developed to improve the separation efficiency; in this chapter, we describe a two-dimensional HPLC separation technique that utilizes a high-pH mobile phase in the first dimension followed by a low-pH mobile phase in the second dimension. This two-dimensional pH-based HPLC approach demonstrates increased separation efficiency of intact proteins and increased proteome coverage when compared to one-dimensional HPLC in the analysis of larger and lower abundance proteoforms.

Quantitative Top-Down Proteomics in Complex Samples Using Protein-Level Tandem Mass Tag Labeling.

Labeling approaches using isobaric chemical tags (e.g., isobaric tagging for relative and absolute quantification, iTRAQ and tandem mass tag, TMT) have been widely applied for the quantification of peptides and proteins in bottom-up MS. However, until recently, successful applications of these approaches to top-down proteomics have been limited because proteins tend to precipitate and "crash" out of solution during TMT labeling of complex samples making the quantification of such samples difficult. In this study, we report a top-down TMT MS platform for confidently identifying and quantifying low molecular weight intact proteoforms in complex biological samples. To reduce the sample complexity and remove large proteins from complex samples, we developed a filter-SEC technique that combines a molecular weight cutoff filtration step with high-performance size exclusion chromatography (SEC) separation. No protein precipitation was observed in filtered samples under the intact protein-level TMT labeling conditions. The proposed top-down TMT MS platform enables high-throughput analysis of intact proteoforms, allowing for the identification and quantification of hundreds of intact proteoforms from Escherichia coli cell lysates. To our knowledge, this represents the first high-throughput TMT labeling-based, quantitative, top-down MS analysis suitable for complex biological samples.

Spray-Capillary-Based Capillary Electrophoresis Mass Spectrometry for Metabolite Analysis in Single Cells.

Single-cell capillary electrophoresis mass spectrometry (CE-MS) is a promising platform to analyze cellular contents and probe cell heterogeneity. However, current single-cell CE-MS methods often rely on offline microsampling processes and may demonstrate low sampling precision and accuracy. We have recently developed an electrospray-assisted device, spray-capillary, for low-volume sample extraction. With the spray-capillary, low-volume samples (pL-nL) are drawn into the sampling end of the device, which can be used directly for CE separation and online MS detection. Here, we redesigned the spray-capillary by utilizing a capillary with a <15 µm tapered tip so that it can be directly inserted into single cells for sample collection and on-capillary CE-MS analysis. We evaluated the performance of the modified spray-capillary by performing single-cell microsampling on single onion cells with varying sample injection times and direct MS analysis or online CE-MS analysis. We have demonstrated, for the first time, online sample collection and CE-MS for the analysis of single cells. This application of the modified spray-capillary device facilitates the characterization and relative quantification of hundreds of metabolites in single cells.

High-throughput hydrogen deuterium exchange mass spectrometry (HDX-MS) coupled with subzero-temperature ultrahigh pressure liquid chromatography (UPLC) separation for complex sample analysis.

Hydrogen deuterium exchange coupled with mass spectrometry (HDX-MS) is a powerful technique for the characterization of protein dynamics and protein interactions. Recent technological developments in the HDX-MS field, such as sub-zero LC separations, large-scale data analysis tools, and efficient protein digestion methods, have allowed for the application of HDX-MS to the analysis of multi protein systems in addition to pure protein analysis. Still, high-throughput HDX-MS analysis of complex samples is not widespread because the co-elution of peptides combined with increased peak complexity after labeling makes peak de-convolution extremely difficult. Here, for the first time, we evaluated and optimized long gradient subzero-temperature ultra-high-pressure liquid chromatography (UPLC) separation conditions for the HDX-MS analysis of complex protein samples such as E. coli cell lysate digest. Under the optimized conditions, we identified 1419 deuterated peptides from 320 proteins at -10 °C, which is about 3-fold more when compared with a 15-min gradient separation under the same conditions. Interestingly, our results suggested that the peptides eluted late in the gradient are well-protected by peptide-column interactions at -10 °C so that peptides eluted even at the end of the gradient maintain high levels of deuteration. Overall, our study suggests that the optimized, sub-zero, long-gradient UPLC separation is capable of characterizing thousands of peptides in a single HDX-MS analysis with low back-exchange rates. As a result, this technique holds great potential for characterizing complex samples such as cell lysates using HDX-MS.

Development of an Online 2D Ultrahigh-Pressure Nano-LC System for High-pH and Low-pH Reversed Phase Separation in Top-Down Proteomics.

The development of novel high-resolution separation techniques is crucial for advancing the complex sample analysis necessary for high-throughput top-down proteomics. Recently, our group developed an offline 2D high-pH RPLC/low-pH RPLC separation method and demonstrated good orthogonality between these two RPLC formats. Specifically, ultrahigh-pressure long capillary column RPLC separation has been applied as the second dimensional low-pH RPLC separation for the improvement of separation resolution. To further improve the throughput and sensitivity of the offline approach, we developed an online 2D ultrahigh-pressure nano-LC system for high-pH and low-pH RPLC separations in top-down proteomics. An online microtrap column with a dilution setup was used to collect eluted proteins from the first dimension high-pH separation and inject the fractions for ultrahigh-pressure long capillary column low-pH RPLC separation in the second dimension. This automatic platform enables the characterization of 1000+ intact proteoforms from 5 µg of intact E. coli cell lysate in 10 online-collected fractions. Here, we have demonstrated that our online 2D pH RP/RPLC system coupled with top-down proteomics holds the potential for deep proteome characterization of mass-limited samples because it allows the identification of hundreds of intact proteoforms from complex biological samples at low microgram sample amounts.

High-throughput quantitative top-down proteomics.

Top-down mass spectrometry (MS) analyzes intact proteins at the proteoform level, which allows researchers to better understand the functions of protein modifications. Recently, top-down proteomics has increased in popularity due to advancements in high-resolution mass spectrometers, increased efficiency in liquid chromatography (LC) separation, and advances in data analysis software. Some unique protein proteoforms, which have been distinguished using top-down MS, have even been shown to exhibit marked variation in biological function compared to similar proteoforms. However, the qualitative identification of a particular proteoform may not be enough to determine the biological relevance of that proteoform. Quantitative top-down MS methods have been notably applied to the study of the differing biological functions of protein proteoforms and have allowed researchers to explore proteomes at the proteoform, rather than the peptide, level. Here, we review the top-down MS methods that have been used to quantitatively identify intact proteins, discuss current applications of quantitative top-down MS analysis, and present new areas where quantitative top-down MS analysis may be implemented.

Spray-Capillary: An Electrospray-Assisted Device for Quantitative Ultralow-Volume Sample Handling.

The analysis of low-volume samples provides valuable insight into complex biological systems. However, the proteomic and metabolomic analysis of low-volume samples remains challenging due to the lack of simple, efficient, and reproducible microsampling techniques. We have developed an electrospray-assisted device for quantitative low-volume sample extraction, referred to here as "Spray-Capillary". Stable electrospray was achieved through a chemically etched tip from a long (e.g., 50 cm) capillary with a conductive sheath flow. This electrospray provided the driving force to quantitatively draw low-volume samples into the capillary. We evaluated the precision and accuracy of sample injection volumes using our spray-capillary as the electrospray voltage, capillary ID, and column length were varied. Our results demonstrate that spray-capillary allows for reproducible and quantitative microsampling with low injection flow rates (as low as 15 pL/s). Furthermore, spray-capillary can be directly coupled with capillary zone electrophoresis (CZE) for separation. Overall, spray-capillary is a simple microsampling device that holds great potential for high-throughput quantitative omics analysis of ultralow-volume samples.

Deep Intact Proteoform Characterization in Human Cell Lysate Using High-pH and Low-pH Reversed-Phase Liquid Chromatography.

Post-translational modifications (PTMs) play critical roles in biological processes and have significant effects on the structures and dynamics of proteins. Top-down proteomics methods were developed for and applied to the study of intact proteins and their PTMs in human samples. However, the large dynamic range and complexity of human samples makes the study of human proteins challenging. To address these challenges, we developed a 2D pH RP/RPLC-MS/MS technique that fuses high-resolution separation and intact protein characterization to study the human proteins in HeLa cell lysate. Our results provide a deep coverage of soluble proteins in human cancer cells. Compared to 225 proteoforms from 124 proteins identified when 1D separation was used, 2778 proteoforms from 628 proteins were detected and characterized using our 2D separation method. Many proteoforms with critically functional PTMs including phosphorylation were characterized. Additionally, we present the first detection of intact human GcvH proteoforms with rare modifications such as octanoylation and lipoylation. Overall, the increase in the number of proteoforms identified using 2DLC separation is largely due to the reduction in sample complexity through improved separation resolution, which enables the detection of low-abundance PTM-modified proteoforms. We demonstrate here that 2D pH RP/RPLC is an effective technique to analyze complex protein samples using top-down proteomics.

Biological Chemistry of Hydrogen Selenide.

There are no two main-group elements that exhibit more similar physical and chemical properties than sulfur and selenium. Nonetheless, Nature has deemed both essential for life and has found a way to exploit the subtle unique properties of selenium to include it in biochemistry despite its congener sulfur being 10,000 times more abundant. Selenium is more easily oxidized and it is kinetically more labile, so all selenium compounds could be considered to be "Reactive Selenium Compounds" relative to their sulfur analogues. What is furthermore remarkable is that one of the most reactive forms of selenium, hydrogen selenide (HSe- at physiologic pH), is proposed to be the starting point for the biosynthesis of selenium-containing molecules. This review contrasts the chemical properties of sulfur and selenium and critically assesses the role of hydrogen selenide in biological chemistry.