Protein extracts obtained from cells or tissues often require removal of interfering substances for the preparation of high-quality protein samples in proteomic analysis. A number of protein extraction methods have been applied to various biological samples. TCA/acetone precipitation and phenol extraction, a common method of protein extraction, is thought to minimize protein degradation and activity of proteases as well as reduce contaminants like salts and polyphenols. However, the TCA/acetone precipitation method relies on the complete pulverization and repeated rinsing of tissue powder to remove the interfering substances, which is laborious and time-consuming. In addition, by prolonged incubation in TCA/acetone, the precipitated proteins are more difficult to re-dissolve. We have described a modified method of TCA/acetone precipitation of plant proteins for proteomic analysis. Proteins of cells or tissues were extracted using SDS-containing buffer, precipitated with equal volume of 20% TCA/acetone, and washed with acetone. Compared to classical TCA/acetone precipitation and simple acetone precipitation, this protocol generates comparable yields, spot numbers, and proteome profiling, but takes less time (ca. 45 min), thus avoiding excess protein modification and degradation after extended-period incubation in TCA/acetone or acetone. The modified TCA/acetone precipitation method is simple, fast, and suitable for proteomic analysis of various plant tissues in proteomic analysis.

/pdf/modified-tca-acetone-precipitation-of-plant-proteins-for-6p43yrdncw.pdf

Modified TCA/acetone precipitation of plant proteins for proteomic analysis

https://febs.onlinelibrary.wiley.com/doi/epdf/10.1016/0014-5793%2893%2981364-6

Enzyme assays: a practical approach

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.

Rapid and Quantitative Protein Precipitation for Proteome Analysis by Mass Spectrometry

Optimisation of protein extraction for in-depth profiling of the cereal grain proteome.

The COVID-19 pandemic caused by the SARS-CoV-2 coronavirus has stretched national testing capacities to breaking points in almost all countries of the world. The need to rapidly screen vast numbers of a country’s population in order to control the spread of the infection is paramount. However, the logistical requirement for reagent supply (and associated cost) of RT-PCR based testing (the current front-line test) have been hugely problematic. Mass spectrometry-based methods using swab and gargle samples have been reported with promise, but have not approached the task from a systematic analysis of the entire diagnostic process. Here, the pipeline from sample processing, the biological characteristics of the pathogen in human biofluid, the downstream bio- and physical-chemistry and the all-important data processing with clinical interpretation and reporting, are carefully compiled into a single high-throughput and reproducible rapid process. Utilizing MALDI-ToF mass spectrometric detection to viral envelope glycoproteins in a systems biology-multidisciplinary team approach, we have achieved a multifaceted clinical MALDI ToF MS screening test, primarily (but not limited to) SARS-CoV-2, with direct application to other future epidemics/pandemics that may arise. The clinical information generated not only includes SARS-CoV-2 coronavirus detection–(Spike protein fragments S1, S2b, S2a peaks), but other respiratory viral infections detected as well as an assessment of generalised oral upper respiratory immune response (elevated total Ig light chain peak) and a measure of the viral immune response (elevated intensity of IgA heavy chain peak). The advantages of the method include; (1) ease of sampling, (2) speed of analysis, and much reduced cost of testing. These features reveal the diagnostic utility of MALDI-ToF mass spectrometry as a powerful and economically attractive global solution

Development of a Clinical MALDI-ToF Mass Spectrometry Assay for SARS-CoV-2: Rational Design and Multi-Disciplinary Team Work

Protein extracts obtained from cells or tissues often need to remove interfering substances for preparing high-quality protein samples in proteomic analysis. A number of protein extraction methods have been applied to various biological samples, especially TCA/acetone precipitation and phenol extraction. TCA/acetone precipitation, as a common method, is thought to minimize protein degradation and proteases activity as well as reduce contaminants like salts and polyphenols. However, the TCA/acetone precipitation relies on the completely pulverizing and repeatedly rinse of tissue powder to remove the interfering substances, which is laborious and time-consuming. In addition, a prolonged incubation in TCA/acetone or acetone can lead to the modifications and degradation of proteins, and the precipitated proteins are more difficult to re-dissolve. We have described a modified TCA/acetone precipitation of plant proteins for proteomic analysis. Proteins of cells or tissues were extracted using SDS-containing buffer, precipitated with equal volume of 20% TCA/acetone, and washed with acetone. Compared to classical TCA/acetone precipitation, this protocol generates comparable yields, spot numbers, and proteome profiling, but takes less time (ca. 45 min), thus avoiding excess protein modification and degradation after extended-period incubation in TCA/acetone or acetone. The modified TCA/acetone precipitation method is simple, fast, and suitable for various plant tissues in proteomic analysis.

https://www.biorxiv.org/content/biorxiv/early/2018/12/01/382317.full.pdf

Modified TCA/acetone precipitation of proteins for proteomic analysis

Recently, technical advances, especially in liquid chromatography (LC) and mass spectrometry (MS), have improved the sensitivity, coverage, reliability, and throughput of proteome analysis (Boersema et al., 2015). Novel proteomics methods, such as targeted proteomics (Marx, 2013), degradomics (Stoehr et al., 2013), structural proteomics (Walzthoeni et al., 2013), chemical proteomics (Rudolf et al., 2013), and microproteomics (Kasuga et al., 2017), are becoming essential tools for in-depth analyses of biological systems and phenomena, such as plant growth, development, and responses to stress factors. The numbers of plant proteins detected using MS-based proteomics remains much lower than expected. For example, the improved maize reference genome contains 39,324 protein-coding genes, with an average of 3.3 transcripts per gene (Jiao et al., 2017), each of which may produce at least several different proteins. Moreover, additional proteins may be synthesized by proteolysis of other existing proteins. To date only 947 reviewed and 169,813 unreviewed maize protein entries have been collected in the UniProtKB (http://www.uniprot.org/uniprot/?query=organism: ‘‘maize’’) (retrieved on Feb 6, 2018). Similarly, analysis of the UniProtKB entries for maize organelle proteins reveals few reviewed proteins compared with a large number of unreviewed entries (Supplementary Table 1). An important reason for this phenomenon is that maize proteomic data has not be curated and collected as the annotation of unreviewed protein entries. Definitely, numerous “missing (hidden) proteins” that are predicted at the transcript level remain unidentified at the protein level in plants. While many factors contribute to missing proteins, one major cause is using inefficient protein extraction methods, especially for hydrophobic membrane proteins and low-abundant proteins (LAPs) (Thelen and Peck, 2007; Libault et al., 2017). Sample quality is critical for the coverage, reliability, and throughput of plant proteomic analysis, although advanced detection approaches (especially LC-MS/MS) can greatly enhance the sensitivity and reliability of protein identification. Here, in view of the current approaches and trends in plant proteomics, we highlight the importance of using multiple protein extraction methods to obtain a more complete picture of plant proteome. Moreover, to promote the identification of more “missing proteins,” we discuss the key aspects of protein extraction methods at the tissue, single-cell, and organelle levels.

Protein Extraction Methods Shape Much of the Extracted Proteomes.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/pbr.12804

Maize mesocotyl: Role in response to stress and deep‐sowing tolerance

Maize is one of the most widely cultivated crops. It accumulates a large quantity of seed storage proteins, which are important for seed development and germination, and contribute to the nutritional quality of seeds. Based on solubility, the storage proteins are divided into albumins (water-soluble), globulins (salt-soluble), prolamins (alcohol-soluble), and glutelins (acid- or alkali-soluble). Maize hybrids are cultivated due to the superior performance of F1 hybrids than that of their parents, a phenomenon known as heterosis. However, the accumulation patterns of seed storage proteins in maize embryos between the hybrids and their parental inbred lines have not been compared. In the present study, two elite inbred lines of China, Zheng 58 and Chang 7-2, and their reciprocal hybrids (Zheng 58 × Chang 7-2 and Chang 7-2 × Zheng 58) were used to explore parental influences on the accumulation patterns of seed storage proteins in maize embryos. For this purpose, we focused on seed salt-soluble proteins (SSPs) in our experiments. The SSPs were selectively extracted from maize mature embryos after extensive removal of water-soluble albumin and separated using two-dimensional gel electrophoresis (2-DE), followed by mass spectrometry analysis. Our results indicated that the 2-DE SSP profiles of hybrids closely resembled those of their maternal parent rather than the paternal parent. In other words, 2-DE SSP profiles of Zheng 58 × Chang 7-2 were more similar those of Zheng 58 whereas such profiles of Chang 7-2 × Zheng 58 were more similar to those of Chang 7-2 although the 2-DE profiles of all four maize types were quite similar. In total, 12 relatively abundant SSPs spots representing five kinds of proteins were identified, of which nine protein spots displayed non-additive accumulation in at least one hybrid. This study provided additional data on dominance and partial dominance effects on maize hybrids embryos. Besides, earlier studies on accumulation profiles of globulin-1 (also known as vicilin), which is one of the most abundant globulins in maize embryos, also support the above results. This study would be helpful in revealing the mechanisms underlying SSPs accumulation patterns in the hybrids.

/pdf/accumulation-profiles-of-embryonic-salt-soluble-proteins-in-10x65p5lch.pdf

Liangjie Niu

Papers

Modified TCA/acetone precipitation of plant proteins for proteomic analysis

Modified TCA/acetone precipitation of proteins for proteomic analysis

Protein Extraction Methods Shape Much of the Extracted Proteomes.

Maize mesocotyl: Role in response to stress and deep‐sowing tolerance

Accumulation Profiles of Embryonic Salt-Soluble Proteins in Maize Hybrids and Parental Lines Indicate Matroclinous Inheritance: A Proteomic Analysis.