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Liquid chromatography–tandem mass spectrometry (LC–MS/MS)-based proteomics is a powerful technique for profiling proteomes of cells, tissues, and body fluids. Typical bottom-up proteomic workflows consist of the following three major steps: sample preparation, LC–MS/MS analysis, and data analysis. LC–MS/MS and data analysis techniques have been intensively developed, whereas sample preparation, a laborious process, remains a difficult task and the main challenge in different applications. Sample preparation is a crucial stage that affects the overall efficiency of a proteomic study; however, it is prone to errors and has low reproducibility and throughput. In-solution digestion and filter-aided sample preparation are the typical and widely used methods. In the past decade, novel methods to improve and facilitate the entire sample preparation process or integrate sample preparation and fractionation have been reported to reduce time, increase throughput, and improve reproducibility. In this review, we have outlined the current methods used for sample preparation in proteomics, including on-membrane digestion, bead-based digestion, immobilized enzymatic digestion, and suspension trapping. Additionally, we have summarized and discussed current devices and methods for integrating different steps of sample preparation and peptide fractionation.
Keywords: proteomics, sample preparation, in-solution digestion, FASP, S-Trap, SP3, LC-MS/MS, automation
Proteomics is an analytical technique that examines protein expression, structures, functions, and interactions in a particular cell, tissue, body fluid, or organism [1,2]. Protein composition and abundance are currently analyzed to identify disease markers or treatment mechanisms, as all changes in proteomes indicate pathological or biological processes [3,4,5]. Over the last two decades, liquid chromatography–tandem mass spectrometry (LC-MS/MS)-based proteomics has been developed as an alternative to time-consuming and labor-intensive gel-based proteomics and immunoassays [6]. LC–MS/MS-based proteomics has high throughput because thousands of peptides and proteins can be analyzed in a short time [7]. Furthermore, it can be automated to improve high-throughput performance, precision, and repeatability [8]. Some liquid-handling workstations, such as Agilent AssayMAP Bravo [9] and Biomek NXP Span-8 [10], can perform most steps of proteomic workflows.
The two analytical procedures frequently used in proteomics are top-down and bottom-up approaches. In top-down proteomics, intact proteins are directly separated and analyzed using LC–MS/MS to identify, characterize, and quantify proteoforms (distinct proteins generated from a particular gene owing to genetic variations), alternative RNA splicing, and post-translational modifications (PTMs) [11,12,13]. Conversely, in bottom-up proteomics, proteins undergo enzymatic proteolysis and the resultant peptides are analyzed and identified. This strategy has been widely used because peptides are easier to separate and identify than proteins [14]. The peptide mixture in bottom-up proteomics comprises thousands of peptides; thus, multidimensional separation is usually performed for in-depth proteome analysis [15].
A typical LC–MS/MS-based proteomic workflow consists of three major steps: sample preparation, protein/peptide separation coupled with MS/MS analysis, and data analysis [16,17]. The two proteomic approaches differ mainly in sample preparation. Top-down proteomics include protein extraction from biological samples and sample purification [18]. However, bottom-up proteomics require additional steps including protein reduction, alkylation, and enzymatic digestion [14]. Each stage of the bottom-up proteomic workflow has tremendously developed over the last two decades. The development of high-performance liquid chromatography (HPLC) instrumentation over the past decade has facilitated proteomic research by simultaneously separating many peptides in a single run [19,20]. Reversed-phase liquid chromatography (RPLC) plays a critical role in protein/peptide separation prior to MS/MS analysis [21]. Other separation mechanisms, such as strong cation exchange chromatography (SCX), strong anion exchange chromatography (SAX), size exclusion chromatography (SEC), and hydrophilic interaction chromatography (HILIC), are frequently combined with RPLC to develop multidimensional separation platforms [15]. Advances in multidimensional separation of proteins/peptides have been demonstrated in various systems, including SCX–RPLC [22,23], SAX–RPLC [24], SEC–RPLC [25,26], HILIC–RPLC [27,28], RPLC–RPLC [29,30], SCX–RPLC–RPLC [31,32,33], SAX–RPLC–RPLC [34], SCX–HILIC–RPLC [35], and RPLC–RPLC–RPLC [36].
The mass spectrometer has also been improved in the past decade with the development of new fragmentation techniques and substantial increases in scan speed and mass accuracy [37,38,39], enabling the first profiling of the human proteome draft in 2014 [37,38]. Some types of mass analyzers frequently used in proteomics are quadrupole, ion-trap, time-of-flight, orbitrap, and Fourier-transform ion cyclotron resonance [39,40]. In recent proteomic studies, data have been analyzed using high-throughput and time-efficient software [41]. Raw MS/MS data are searched against databases to identify and quantify peptides and proteins using search engines such as X!Tandem [42], Mascot [43], Sequest [44], Comet [45], Maxquant [46], Byonic [47], MSFragger [48], and Open-pFind [49]. The results are further subjected to statistical tests to identify differentially expressed proteins (DEPs), enrichment analysis to determine biological relevance, and network analysis to visualize protein–protein interactions and protein groups [41].
Apart from these stages, sample preparation remains a difficult task and the main challenge of bottom-up proteomics with laborious steps [50]. Generally, sample preparation aims to create a less complex peptide mixture that is suitable for analysis. It requires pre-fractionation; depletion of most unnecessarily abundant proteins; removal of DNA, lipids, and small metabolites; and sample clean-up from impurities (salts and remaining solid particles) [51]. Therefore, typical sample preparation processes for bottom-up proteomics usually include lysis/homogenization, protein extraction/precipitation, reduction, alkylation, enzymatic digestion, fractionation, and desalting ( Figure 1 ). Sample preparation is an essential stage affecting the overall efficiency of proteomic studies. However, it is error-prone and has low reproducibility and throughput [52]. The early stages of proteomics used in-gel sample preparation [53]. Gel-free sample preparation has been developed in the past decade and is widely used in proteomic studies. Typical strategies include in-solution digestion (ISD), filter-aided sample preparation (FASP) [54], suspension trapping (S-Trap) [55], and single-pot solid-phase-enhanced sample preparation (SP3) [56]. Many groups have reported novel methods to improve and facilitate the entire sample preparation process or some of its steps to reduce time, increase throughput, and improve reproducibility. Single-cell proteomics has been developed in the past decades to handle samples with low protein amounts [57]. Typical single-cell proteomic approaches are: nanodroplet processing in one pot for a trace sample (nanoPOTS) [58]; nanoliter-scale oil-air-droplet (OAD) chip [59]; an integrated device for single-cell analysis (iPAD-1) [60]; digital microfluidic isolation of single cells for -omics (DISCO) [61]; and integrated spectral library-based single-cell proteomics [62]. However, this review does not cover single-cell proteomics due to their unique characteristics. In this review, we have summarized the current methods for preparing different proteomic sample types. In addition, we have presented and discussed recent developments to enhance the sample preparation process and integrate different sample preparation steps as well as their applications to biological samples.
Sample preparation in bottom-up proteomics.
