Proteomic Sample Preparation Research Articles

Defining Proteomic Signatures to Predict Multidrug Persistence in Pseudomonas aeruginosa.

Bacterial persisters are difficult to eradicate because of their ability to survive prolonged exposure to a range of different antibiotics. Because they often represent small subpopulations of otherwise drug-sensitive bacterial populations, studying their physiological state and antibiotic stress response remains challenging. Sorting and enrichment procedures of persister fractions introduce experimental biases limiting the significance of follow-up molecular analyses. In contrast, proteome analysis of entire bacterial populations is highly sensitive and reproducible and can be employed to explore the persistence potential of a given strain or isolate. Here, we summarize methodology to generate proteomic signatures of persistent Pseudomonas aeruginosa isolates with variable fractions of persisters. This includes proteome sample preparation, mass spectrometry analysis, and an adaptable machine learning regression pipeline. We show that this generic method can determine a common proteomic signature of persistence among different P. aeruginosa hyper-persister mutants. We propose that this approach can be used as diagnostic tool to gauge antimicrobial persistence of clinical isolates.

Identification of Deamidated Peptides in Cytokine-Exposed MIN6 Cells through LC-MS/MS Using a Shortened Digestion Time and Inspection of MS2 Spectra.

Enzymatic deamidation, the conversion of glutamine (Gln) into glutamic acid (Glu) residues, mediated by tissue transglutaminase enzymes, can provoke autoimmunity by generating altered self-epitopes, a process well-known in celiac disease and more recently also described in type 1 diabetes (T1D). To identify deamidated proteins, liquid chromatography-tandem mass spectrometry is the method of choice. However, as nonenzymatic deamidations on asparagine (Asn) and to a minor extent on Gln are frequently induced in vitro during proteomics sample preparation, the accurate detection of in vivo deamidation can be hampered. Here we report on the optimization of a method to reduce in vitro generated deamidation by 70% using improved trypsin digestion conditions (90 min/pH 8). We also point to the critical importance of manual inspection of MS2 spectra, considering that only 55% of the high quality peptides with Gln deamidation were assigned correctly using an automated search algorithm. As proof of principal, using these criteria, we showed a significant increase in levels of both Asn and Gln deamidation in cytokine-exposed murine MIN6 β-cells, paralleled by an increase in tissue transglutaminase activity. These findings add evidence to the hypothesis that deamidation is occurring in stressed β-cell proteins and can be involved in the autoimmune process in T1D.

Phosphoproteome and Proteome Sample Preparation from Mouse Tissues for Circadian Analysis.

Recent advances in mass spectrometry (MS)-based quantitative proteomics now allow the identification and quantification of deep proteomes and post-translational modifications (PTMs) in relatively short times. Therefore, in the last few years, this technology has proven successful in the circadian field to characterize temporal oscillations of the proteome and more recently PTMs in cellular systems and in tissues. In this chapter, we describe a robust and simple protocol, based on the EasyPhos workflow, to enable preparation of large number of proteomes and phosphoproteomes from mouse tissues for MS-based quantitative analysis. We additionally discuss computational methods to analyze proteome and phosphoproteome time series to determine circadian oscillations.

Adapting a Low-Cost and Open-Source Commercial Pipetting Robot for Nanoliter Liquid Handling.

Low-volume liquid handling capabilities in bioanalytical workflows can dramatically improve sample processing efficiency and reduce reagent costs, yet many commercial nanoliter liquid handlers cost tens of thousands of dollars or more. We have successfully adapted a low-cost and open-source commercial pipetting robot, the Opentrons OT-1, to accurately aspirate and dispense nanoliter volumes. Based on fluorescence measurements, the modified OT-1 was able to reproducibly transfer 50 nL of water with less than 3% measurement error and 5% coefficient of variation (CV). For 15 nL transfers, the volume measurements indicated less than 4% error and 4% CV. We applied this platform to the preparation of low-nanogram proteomic samples for liquid chromatography-mass spectrometry analysis, demonstrating that the modified OT-1 is an effective platform for nanoliter liquid handling. At a total materials cost of less than $6000, including the commercial liquid handler and all modifications, this system is also far less expensive than other platforms with similar capabilities, placing automated nanoliter handling within reach of a far broader scientific community.

Proximity labeling in mammalian cells with TurboID and split-TurboID.

This protocol describes the use of TurboID and split-TurboID in proximity labeling applications for mapping protein-protein interactions and subcellular proteomes in live mammalian cells. TurboID is an engineered biotin ligase that uses ATP to convert biotin into biotin-AMP, a reactive intermediate that covalently labels proximal proteins. Optimized using directed evolution, TurboID has substantially higher activity than previously described biotin ligase-related proximity labeling methods, such as BioID, enabling higher temporal resolution and broader application in vivo. Split-TurboID consists of two inactive fragments of TurboID that can be reconstituted through protein-protein interactions or organelle-organelle interactions, which can facilitate greater targeting specificity than full-length enzymes alone. Proteins biotinylated by TurboID or split-TurboID are then enriched with streptavidin beads and identified by mass spectrometry. Here, we describe fusion construct design and characterization (variable timing), proteomic sample preparation (5-7 d), mass spectrometric data acquisition (2 d), and proteomic data analysis (1 week).

Cysteine alkylation methods in shotgun proteomics and their possible effects on methionine residues

In order to optimize sample preparation for shotgun proteomics, we compared four cysteine alkylating agents: iodoacetamide, chloroacetamide, 4-vinylpyridine and methyl methanethiosulfonate, and estimated their effects on the results of proteome analysis. Because alkylation may result in methionine modification in vitro, proteomics data were searched for methionine to isothreonine conversions, which may mimic genomic methionine to threonine substitutions found in proteogenomic analyses. We found that chloroacetamide was superior to the other reagents in terms of the number of identified peptides and undesirable off-site reactions. Among the reagents evaluated, iodoacetamide increased the rate of methionine-to-isothreonine conversion, especially if the sample was prepared in gel. The presence of proline following methionine in a protein sequence increased the modification rate as well. Generally, the methionine-to-isothreonine conversion events were relatively rare, but should be taken into account in proteogenomic studies when searching for single nucleotide polymorphism events at the protein level. Additionally, we have evaluated other methionine modifications, such as oxidation and carbamidomethylation. We found that carbamidomethylation may affect up to 80% of peptides containing methionine under the condition of iodoacetamide alkylation. In this case, carbamidomethylation of methionine is more common than oxidation and should be accounted for as a variable modification during proteomic search. SignificanceOne of the most trending questions in bottom-up proteomics is the depth of proteome profiling, in other words, the coverage of proteins by identified tryptic peptides. In proteogenomics, where the identification of a single peptide, e.g. bearing an amino acid substitution, may be of interest, high sequence coverage is especially important. Chemical modifications during sample preparation may mimic biologically significant coding mutations at the proteome level. A typical example of such modification is methionine to isothreonine conversion during alkylation, which mimics methionine to threonine substitution in protein sequences due to respective genomic mutations. Therefore, the studies on the proper selection of alkylating reagents which balance the cysteine alkylation efficiency and the extent of methionine conversion upon conventional proteomic sample preparation workflow are crucial for the outcome of proteogenomic analyses and should present a general interest for the proteomic community.

Adding polyvinylpyrrolidone to low level protein samples significantly improves peptide recovery in FASP digests: An inexpensive and simple modification to the FASP protocol

Filter-aided sample preparation (FASP) remains a popular choice for proteomic sample preparation, particularly for its ability to produce a 'clean' peptide sample clear of large molecule contaminants. However, sample loss continues to be a problem particularly for sample inputs that contain less than ten micrograms of protein. Here, we describe that the simple addition of a polymer, polyvinylpyrrolidone-40 (PVP-40) to the protein sample prior to FASP digest significantly improves peptide recovery and identifications, especially with lower level sample inputs. PVP-FASP produces clean samples which required no additional sample clean-up prior to nanoLC-MS analysis. In addition, PVP-FASP is compatible with other FASP modifications, including the use of sodium deoxycholate (DOC) to improve trypsin digestion. SIGNIFICANCE: Simple modification to FASP procedure improves sample recovery during proteomic digests in SDS, improving peptide identifications and median peptide intensity.

Formaldehyde Derivatization: An Unexpected Side Reaction During Filter-Aided Sample Preparation.

The filter-aided sample preparation (FASP) method has been commonly used for proteomic sample preparation due to its high efficiency in removing impurities. Herein, we report an overlooked +12 Da side modification during FASP method using Microcon spin filters. We confirmed that the side modification is caused by formaldehyde released from the spin filter and found that the side modification leads to 10.5% and 9.5% loss in proteome-level peptide and protein identification, respectively. We evaluated different pretreatment procedures to reduce the side reaction. Furthermore, on the basis of the evaluation results of different brands of spin filters, we recommend Nanosep spin filters for different proteomic studies, especially for amine-labeling proteomic studies. Our results would benefit researchers employing the spin filters to improve their results and also help spin filter manufacturers to improve the product quality. Data are available via ProteomeXchange with identifier PXD018737.

Optimisation of urine sample preparation for shotgun proteomics

AbstractUrine reflects the renal function and urinary and kidney systems, but it may also reflect the presence of cancer in other parts of the body. Urine also has potential for providing prognostic information during therapeutic treatments thanks to non-invasive monitoring. A quick and reproducible protein purification procedure is essential to allow data comparison between proteomic studies in urine biomarker discovery. The article describes a simple, reproducible and cheap sample preparation procedure with a maximum protein yield (400 µg) obtained from only 10 mL of urine utilising cut-off filter desalting and digestion. The reported procedure removes yellowish background coloration residues and thus prevents the errors in spectrophotometric protein concentration determination. Different extraction solvents used in the presented procedure point to the possibility of partial elimination of abundant proteins (albumin and keratin family), as well as to the improvement of the sequence coverage of proteins identified, which helps to reveal changes in the urinary proteome. With this workflow, proteins can be easily obtained on standard laboratory equipment within 3 h. Data are available via ProteomeXchange with identifier PXD019738.

AutoProteome Chip System for Fully Automated and Integrated Proteomics Sample Preparation and Peptide Fractionation.

With recent advances in LC-MS systems, current MS-based proteomics has an increasing need for automated, high-throughput sample preparation with neglectable sample loss. In this study, we developed a microfluidic system for fully automated proteomics sample preparation. All of the required proteomics sample preparation steps for both protein digestion and peptide fractionation are fully integrated into a disposable plastic chip device (named AutoProteome Chip). The AutoProteome Chip packed with mixed-mode ion exchange beads and C18 membrane in tandem could be fabricated with very low cost and high stability in organic reagents. Benefiting from its low backpressure, the AutoProteome Chip could be precisely driven by gas pressure, which could be easily multiplexed. As low as 2 ng of standard protein BSA could be trapped into the AutoProteome chip and processed within 2 h. Fully automated processing of 10 μg of protein extracts of HEK 293T cells achieved more than 97% of digestion efficiency with missed cleavage less than 2 and comparable performance with conventional approaches. More than 4700 proteins could be readily identified within 80 min of LC-MS analysis with good label-free quantification performance (Pearson correlation coefficient >0.99). Furthermore, deep proteome profiling by integrated high-pH RP fractionation in the same AutoProteome Chip resulted in more than 7500 proteins being identified from only 20 μg of protein extracts of HEK 293T cells and comparable reprodicibility as single-shot analysis. The AutoProteome Chip system provided a valuable prototype for developing a fully automated proteome analysis workflow and for proteomic applications with high demand for processing throughput, reproducibility, and sensitivity.

Ultrasonic-Based Filter Aided Sample Preparation as the General Method to Sample Preparation in Proteomics

We propose a new high-throughput ultrafast method for large-scale proteomics approaches by speeding up the classic filter aided sample preparation protocol, FASP, from overnight to 2.5 h. Thirty-six samples can be treated in 2.5 h, and the method is scalable to 96-well plate-based pipelines. After a modification of the FASP-tube, the steps of protein reduction, protein alkylation, and protein digestion of complex proteomes are done in just 5.25 min, each one under the effects of an ultrasonic field (7 cycles: 30 s on and 15 s off). The new method was compared to the standard overnight digestion FASP protocol, and no statistical differences were found for more than 92.4%, 92%, and 93.3% of the proteins identified by studying the proteome of E. coli, mouse brain, and mouse liver tissue samples, respectively. Furthermore, the successful relative label-free quantification of four spiked proteins in E. coli samples, BSA, β-lactoglobulin, α-casein, and α-lactalbumin, was achieved, using either the ultrasonic-based FASP protocol or the classic overnight one. The new US-FASP method matches the analytical minimalism rules as time, cost, sample requirement, reagent consumption, energy requirements, and production of waste products are reduced to a minimum while maintaining high sample throughput in a robust manner as all of the advantages of the filter aided sample preparation protocol are maintained.

Profiling the ‘deamidome’ of complex biosamples using mixed-mode chromatography-coupled tandem mass spectrometry

Deamidation is a spontaneous degenerative protein modification (DPM) that disrupts the structure and function of both endogenous proteins and various therapeutic agents. While deamidation has long been recognized as a critical event in human aging and multiple degenerative diseases, research progress in this field has been restricted by the technical challenges associated with studying this DPM in complex biological samples. Asparagine (Asn) deamidation generates L-aspartic acid (L-Asp), D-aspartic acid (D-Asp), L-isoaspartic acid (L-isoAsp) or D-isoaspartic acid (D-isoAsp) residues at the same position of Asn in the affected protein, but each of these amino acids displays similar hydrophobicity and cannot be effectively separated by reverse phase liquid chromatography. The Asp and isoAsp isoforms are also difficult to resolve using mass spectrometry since they have the same mass and fragmentation pattern in MS/MS. Moreover, the 13C peaks of the amidated peptide are often misassigned as monoisotopic peaks of the corresponding deamidated peptides in protein database searches. Furthermore, typical protein isolation and proteomic sample preparation methods induce artificial deamidation that cannot be distinguished from the physiological forms. To better understand the role of deamidation in biological aging and degenerative pathologies, new technologies are now being developed to address these analytical challenges, including mixed mode electrostatic-interaction modified hydrophilic interaction liquid chromatography (emHILIC). When coupled to high resolution, high accuracy tandem mass spectrometry this technology enables unprecedented, proteome-wide study of the 'deamidome' of complex samples. The current article therefore reviews recent advances in sample preparation methods, emHILIC-MS/MS technology, and MS instrumentation / data processing approaches to achieving accurate and reliable characterization of protein deamidation in complex biological and clinical samples.

A Plasma Sample Preparation for Mass Spectrometry using an Automated Workstation.

Sample preparation for mass spectrometry analysis in proteomics requires enzymatic cleavage of proteins into a peptide mixture. This process involves numerous incubation and liquid transfer steps in order to achieve denaturation, reduction, alkylation, and cleavage. Adapting this workflow onto an automated workstation can increase efficiency and reduce coefficients of variance, thereby providing more reliable data for statistical comparisons between sample types. We previously described an automated proteomic sample preparation workflow1. Here, we report the development of a more efficient and better controlled workflow with the following advantages: 1) The number of liquid transfer steps is reduced from nine to six by combining reagents; 2) Pipetting time is reduced by selective tip pipetting using a 96-position pipetting head with multiple channels; 3) Potential throughput is increased by the availability of up to 45 deck positions; 4) Complete enclosure of the system provides improved temperature and environmental control and reduces the potential for contamination of samples or reagents; and 5) The addition of stable isotope labeled peptides, as well as β-galactosidase protein, to each sample makes monitoring and quality control possible throughout the entire process. These hardware and process improvements provide good reproducibility and improve intra-assay and inter-assay precision (CV of less than 20%) for LC-MS based protein and peptide quantification. The entire workflow for digesting 96 samples in a 96-well plate can be completed in approximately 5 hours.

Rapid and Quantitative Protein Precipitation for Proteome Analysis by Mass Spectrometry.

Protein precipitation is a common front-end preparation strategy for proteome analysis, as well as other applications (e.g., protein depletion for small molecule analysis, bulk commercial preparation of protein). Highly variable conditions used to precipitate proteins, ranging in solvent type, strength, time, and temperature, reflect inconsistent and low recovery. As a consequence, incomplete proteome coverage diminishes the utility of precipitation for proteome sample preparation ahead of mass spectrometry. We herein investigate and optimize the conditions affecting protein recovery through precipitation using acetone at a defined ionic strength. By increasing the salt concentration and incubation temperature with 80% acetone, we show that rapid (2 min) precipitation provides consistently high protein recovery (98 ± 1%) of complex proteome extracts. Rapid precipitation is also applicable to isolate dilute proteins starting as low as 1 μg mL-1. Furthermore, analysis of the protein pellet by bottom-up mass spectrometry (MS) reveals unbiased recovery of all proteins with respect to molecular weight, isoelectric point (pI), and hydrophobicity. Our robust strategy to isolate proteins maximizes recovery and throughput, exploiting the analytical advantages of precipitation over alternative techniques. Data are available via ProteomeXchange with identifier PXD015674.

Evaluation and minimization of nonspecific tryptic cleavages in proteomic sample preparation.

In this study, protein extraction and trypsin digestion conditions were extensively evaluated using the less-complex Escherichia coli lysates to improve the sensitivity of detecting low-abundance nonspecific peptides by liquid chromatography/tandem mass spectrometry. Trypsin digestion buffers and digestion times were proved to have a significant effect on nonspecific cleavages. The triethylammonium bicarbonate buffer induces significantly lower nonspecific cleavages than the other two buffers, but a freshly prepared urea solution does not induce more than sodium dodecyl sulfate. Because prolonged trypsin digestion resulted in a considerable number of nonspecific cleavages, an optimized 2-h protocol was developed with 45.2% less semispecific tryptic peptides but 18.5% more unmodified peptides identified than the commonly used 16-h protocol. The significant decrease in nonspecific cleavages and artificial modifications improves the accuracy of protein quantification and the identification of low-abundance proteins, and it is especially useful for studying protein posttranslational modifications. For trypsin digestion, the proposed 2-h protocol can potentially be a replacement for the traditional 16-h protocol.

Miniaturized Filter-Aided Sample Preparation (MICRO-FASP) Method for High Throughput, Ultrasensitive Proteomics Sample Preparation Reveals Proteome Asymmetry in Xenopus laevis Embryos.

We report a miniaturized filter aided sample preparation method (micro-FASP) for low-loss preparation of submicrogram proteomic samples. The method employs a filter with ∼0.1 mm2 surface area, reduces the total volume of reagents to <10 μL, and requires only two sample transfer steps. The method was used to generate 25 883 unique peptides and 3069 protein groups from 1000 MCF-7 cells (∼100 ng protein content), and 13 367 peptides and 1895 protein groups were identified from 100 MCF-7 cells (∼10 ng protein content). Single blastomeres from Xenopus laevis embryos at the 50-cell stage (∼200 ng yolk free protein/blastomere) generated 20 943 unique peptides and 2597 protein groups; the proteomic profile clearly differentiated left and right blastomeres and provides strong support for models in which this asymmetry is established early in the embryo. The parallel processing of 12 samples demonstrates reproducible label free quantitation of 1 μg protein homogenates.

In-Line Sample Processing System with an Immobilized Trypsin-Packed Fused-Silica Capillary Tube for the Proteomic Analysis of a Small Number of Mammalian Cells.

Omics analysis at single-cell resolution has helped to demonstrate the shaping of cellular heterogeneity on the basis of the expression of various molecules. However, in-depth proteomic analysis of low-quantity samples has remained challenging because of difficulties associated with the measurement of large numbers of proteins by shotgun proteomics using nanoflow liquid chromatography tandem mass spectrometry (nano-LC/MS/MS). To meet such a demand, we developed a method called in-line sample preparation for efficient cellular proteomics (ISPEC) in which cells were captured, directly lysed, and digested with immobilized trypsin within fused-silica capillaries. ISPEC minimized sample loss during the sample preparation processes with a relatively small number of mammalian cells (<1000 cells) and improved the stability and efficiency of digestion by immobilized trypsin, compared to a conventional preparation method. Using our optimized ISPEC method with nano-LC/MS/MS analysis, we identified 1351, 351, and 60 proteins from 100 cells, 10 cells, and single cells, respectively. The linear response of the signal intensity of each peptide to the introduced cell number indicates the quantitative recovery of the proteome from a very small number of cells. Thus, our ISPEC strategy facilitates quantitative proteomic analysis of small cell populations.

‘Shotgun’ proteomic analyses without alkylation of cysteine

It is a common belief that reduction of disulfide bridges and alkylation of thiols in proteins are indispensable steps in proteomic sample preparation. Since this chemical procedure is often incomplete and prone to side reactions we reexamined its importance. We found that reduction and alkylation do not increase the depth of analysis and quality of proteomic quantification and therefore these steps are not essential in ’shotgun’-type investigations of proteomes. Moreover, we found that compared to a standard procedure using iodoacetamide for thiol-alkylation, sample preparation under conditions protecting thiols from oxidation improves quality of peptides and allows identifying of 10–20% more peptides and proteins. Excluding thiol-alkylation from proteomic sample preparation shortens the workflows and decreases the probability of biases resulting from occurrence of artificially modified peptides.

Investigating Monoliths (Vinyl Azlactone-co-Ethylene Dimethacrylate) as a Support for Enzymes and Drugs, for Proteomics and Drug-Target Studies.

Prior to mass spectrometry, on-line sample preparation can be beneficial to reduce manual steps, increase speed, and enable analysis of limited sample amounts. For example, bottom-up proteomics sample preparation and analysis can be accelerated by digesting proteins to peptides in an on-line enzyme reactor. We here focus on low-backpressure 100 μm inner diameter (ID) × 160 mm, 180 μm ID × 110 mm or 250 μm ID × 140 mm vinyl azlactone-co-ethylene dimethacrylate [poly(VDM-co-EDMA)] monoliths as supports for immobilizing of additional molecules (i.e., proteases or drugs), as the monolith was expected to have few unspecific interactions. For on-line protein digestion, monolith supports immobilized with trypsin enzyme were found to be suited, featuring the expected characteristics of the material, i.e., low backpressure and low carry-over. Serving as a functionalized sample loop, the monolith units were very simple to connect on-line with liquid chromatography. However, for on-line target deconvolution, the monolithic support immobilized with a Wnt pathway inhibitor was associated with numerous secondary interactions when exploring the possibility of selectively trapping target proteins by drug-target interactions. Our initial observations suggest that (poly(VDM-co-EDMA)) monoliths are promising for e.g., on-line bottom-up proteomics, but not a “fit-for-all” material. We also discuss issues related to the repeatability of monolith-preparations.

Sample Preparation by Easy Extraction and Digestion (SPEED) - A Universal, Rapid, and Detergent-free Protocol for Proteomics Based on Acid Extraction.

The main challenge of bottom-up proteomic sample preparation is to extract proteomes in a manner that enables efficient protein digestion for subsequent mass spectrometric analysis. Today's sample preparation strategies are commonly conceptualized around the removal of detergents, which are essential for extraction but strongly interfere with digestion and LC-MS. These multi-step preparations contribute to a lack of reproducibility as they are prone to losses, biases and contaminations, while being time-consuming and labor-intensive. We report a detergent-free method, named Sample Preparation by Easy Extraction and Digestion (SPEED), which consists of three mandatory steps, acidification, neutralization and digestion. SPEED is a universal method for peptide generation from various sources and is easily applicable even for lysis-resistant sample types as pure trifluoroacetic acid (TFA) is used for highly efficient protein extraction by complete sample dissolution. The protocol is highly reproducible, virtually loss-less, enables very rapid sample processing and is superior to the detergent/chaotropic agent-based methods FASP, ISD-Urea and SP3 for quantitative proteomics. SPEED holds the potential to dramatically simplify and standardize sample preparation while improving the depth of proteome coverage especially for challenging samples.