I believe that children's shoes that have read Bo Xiaoyuan's previous articles should find that there are many places in the article that have appeared in the mass spectrum. However, in the past few days, Xiaoyuan seems to have never introduced mass spectra to everyone in a serious way, but the light of mass spectrometry is too dazzling, so this issue xiaoyuan intends to officially introduce mass spectrometry to everyone!
Mass spectrometry definition
Mass spectrometry is an application technology based on the theory of atomic, molecular ionization and ion optics, through mass spectrometry analysis can obtain inorganic, organic and biological molecule molecular weight and molecular structure, can be a variety of complex mixtures of various components for quantitative or qualitative analysis. Mass spectrometry technology is actually the application of the classic "motion of charged particles in magnetic fields" problem in high school physics textbooks. In simple terms, it is to put a complex mixture in a mass spectrometry system, decompose it into atoms and molecules, and then inject the ionized atoms and molecules into the electromagnetic field. Since the mass and charge ratios of different charged particles are different, and the deflection time is also different, the mass spectrometer can convert these time and position information into optical data, which is displayed in the form of a mass spectrogram, and the various components of the complex mixture can be intuitively observed. The abscissa of the mass spectrogram is the mass-to-charge ratio (m/z) and the ordinate is the ion intensity. The concept may sound more dry and difficult to understand, but it doesn't matter, we mainly talk about its application today, see what it can do, and then slowly understand the principle later, maybe it is not so difficult!
Protein identification
In proteomics, protein identification is achieved almost entirely by mass spectrometry, which can identify any protein with a known genomic sequence. In addition, mass spectrometry methods are most commonly used to analyze polypeptides rather than full-length proteins, and many times we will see mass spectrometry in conjunction with other techniques to identify proteins! It may be a bit abstract to say so, but there is an experiment that everyone should have heard of - IP-MS (immunoprecipitation - mass spectrometry combined technology), which has appeared in the previous articles, in order to deepen everyone's understanding of the technology, Bo Xiaoyuan found relevant experimental cases to explain to everyone again.
In "Leucine-rich repeat extensin proteins regulate plant salt tolerance in Arabidopsis," the authors used 35S::LRX3-YFP-HA, 35S::LRX4-YFP-HA, and 35S:: LRX5-YFP-HA transgenic plants were subjected to immunoprecipitation-mass spectrometry (IP-MS) analysis to identify potential interacting proteins in LRXs. Transgenic plants expressing 35S::GFP were also used as controls. It was found that four phylogenetically associated RALF polypeptides: RALF22, RALF23, RALF24, and RALF31 were identified in IP-MS samples of LRX transgenic plants, but were not found in control samples (Figure 1). In the later experiment, I think most of the friends already know what it is. That's right! Is the use of peer-to-peer verification of protein interaction method to IP-MS identification of the LRX interaction protein to verify, this routine believes that everyone is already familiar with can no longer be familiar with, so the unclear small partner must write it down!
Figure 1 Polypeptides of LRX and RALF proteins identified in IP-MS detection (Zhao et al., 2018). Proteins were extracted from 35S::LRX3-YFP-HA, 35S::LRX4-YFP-HA, and 35S::LRX5-YFP-HA transgenic plants. Control was given to transgenic plants that overexpressed no-loaded GFP. The isolated proteins are incubated overnight with anti-GFP antibodies. Immunoprecipitated samples are analyzed by mass spectrometry. The number of peptides identified per protein is shown, with a dash "-" indicating that the peptide has not been identified.
If we want to study what an interoprotection protein of an unknown protein is, in addition to thinking of experiments with yeast double heterosis, IP-MS is also the method we should think of. IP-MS not only yields similar results to yeast double heterozygous libraries, but also offers the following advantages:
1. IP-MS screening is the interaction of proteins in the natural state (without destroying the structure of proteins), which can better reflect the real situation in the organism to a certain extent;
2, do not worry about the self-activating effect of bait proteins like the yeast double heterogeneous sieve library.
Therefore, in the future, when studying the interaction protein of an unknown protein, you can first consider IP-MS!
Mass spectrometry quantitative analysis
In addition to the initial identification of phenotypic expression and protein properties, a key parameter in proteomic analysis is the ability to quantify the protein of interest. About two decades ago, proteomics was largely a qualitative discipline. The result of a typical proteomics experiment is to identify a list of proteins in a given tissue or protein complex without any further information about abundance, distribution, or stoichiometry. In contrast, quantitative strategies at that time had been widely used in gene expression analysis for microarrays or quantitative PCR, especially in plants, where large-scale genomics assays provided new insights into many aspects of plant development and physiology. However, enzyme reactions and signaling pathways ultimately depend on the activity of the protein. The amount of protein is regulated by protein synthesis and degradation and may not have much to do with transcriptional regulation (Piques et al., 2022). In addition, post-translational modifications, isomers, and splicing variants cannot be obtained by analyzing the abundance of transcripts alone. Therefore, in order to explain some biological phenomena, protein quantification is necessary. The advent of mass spectrometry provides a technical framework for proteomic complexity analysis.
Quantitative proteomics can be divided into relative quantification and absolute quantification. Relative quantification is designed to study differences in proteome expression under different conditions, while absolute quantification refers to the specific level of expression of obtained proteins. Absolute quantification requires the use of standardized reference samples. Xiaoyuan lists some common mass spectrometry quantitative methods in the following table, everyone is interested in in-depth understanding, here is not a specific introduction Oh!
Table 1 Overview of the technical parameters of different workflows of proteomics
concentrate:
1 Absolute quantification can only be achieved by making a relative comparison with a labeled known standard.
2 Absolute quantification can only be performed by empirical characteristics and inverse calculations using the molecular weight and total protein amount of the proteins in the sample.
MS2: Mass spectrometry detects all ions produced after the peptide ions are broken. Fragment ion scanning is also known as AN MS2 scan or MS/MS scan.
Mass spectrometry determines post-translational modification of proteins
Post-translational modifications (PTMs) are processes that change the properties of proteins by proteolysis and cleavage or by covalently adding modified groups to one or more amino acids. Describing the function of a protein solely in terms of changes in abundance is very limited to understanding the proteome, as the activity of many proteins is regulated by PTMs, which may not accurately reflect changes in protein abundance. This is why many mass spectrometry techniques used to characterize and quantify proteins and peptides in the proteome range have been applied to characterize proteins that are modified after translation.
There are many types of PTMs, including phosphorylation, ubiquitination, acetylation, glycosylation, etc. Mass spectrometry is considered a key technique for protein modification analysis because it can provide general information about protein modification without first knowing the location of the modification site. Top-down, center-up, and bottom-up approaches are three main strategies for MS-based PTM analysis.
For mass spectrometry protein quantification and mass spectrometry to determine the post-translational modification of translation proteins, Xiao Yuan first uses an example to give you a simple explanation, and then explains this part in detail later, because this part is also a big project!
Here comes the chestnuts:
The model plant Arabidopsis thaliana has not only changed our understanding of plant biology, but has also influenced many other areas of the life sciences. The findings from Arabidopsis thaliana also provide ideas for the study of important agronomic traits of crops. Arabidopsis's genome was sequenced 20 years ago, and since then hundreds of natural variants have been analyzed at the genomic and epigenome level. In contrast, the Arabidopsis proteome, as the primary performer of most life processes, is far less comprehensive in its characteristics. To address this gap, in the article "Mass-spectrometry-based draft of the Arabidopsis proteome," the authors used state-of-the-art mass spectrometry and RNA sequencing (RNA-seq) analysis to obtain the first integrated proteome, phosphorus proteome, and transcriptome atlas of Arabidopsis known to us. This rich molecular resource can be used to explore the function of a single protein or entire pathways across several omics levels.
Figure 2 Organization chart and multi-set datasets (Mergner et al., 2020). a. Schematic diagram of the tissue samples analyzed, colored according to morphological groupings (abbreviation definition in b): flowers (light gray); seeds (dark brown); pollen (yellow); stems (dark green); leaves (light green); roots (dark gray); fruits (light brown); callus (red); cell culture (blue). b. Number of proteins, P-sites, and transcriptional levels recognized by all tissues (n=1 measurement). The dotted line indicates the number of core proteins, P-sites, or transcripts detected in all tissues. Tissue-enhanced proteins or transcripts are labeled with a darker color. P-sites with high confidence amino acid localization (Class I sites; localization probabilities greater than 0.75) are represented in pink; ambiguous site localization is indicated in purple. The number of P-sites specifically detected in a tissue is represented by circles. c. The total number and overlap of gene loci identified in the transcriptome, proteome, and phosphorylated proteome dataset compared to Araport11 , and the ratio of the total number of P- loci and class I loci identified (right) compared to Araport11.
As can be seen through this example, mass spectrometry can not only obtain quantitative data on protein levels, but also identify phosphorylation sites to obtain phosphorylated proteome datasets. Compared with RNA-seq, mass spectrometry is not much more powerful, in addition to identifying phosphorylation, other protein post-translational modifications can also be identified by mass spectrometry, if you want to know more, please pay attention to the late article of Bo Xiaoyuan!
Mass spectrometry and metabolism
Metabolomics is a relatively new field of study, however, since Oliver and his collaborators introduced the term metabolome in 1998, publications on the subject have grown exponentially. Plants are very rich in metabolites compared to animals; the total number of metabolites in plants is estimated at 200,000 or more (K. Oksman-Caldentey and K. Saito, 2005). Because of this, plant metabolomics has been widely used in many fields, such as for taxonomy or biochemistry, fingerprint analysis of genes of interest or ecotype, comparison of mutants and transgenic plants with their wild types, the influence of external stimuli on metabolite spectra, plant-herbivores interactions, developmental processes, quality control of medicinal herbs, and determination of medicinal plant activity (Wolfender et al., 2013). Since the applications of plant metabolomics are very diverse, the techniques used to analyze plant metabolomics are also diverse. Combined methods, such as mass spectrometry and gas chromatography or liquid chromatography (GC-MS and LC-MS), direct injection mass spectrometry (DIMS), nuclear magnetic resonance (NMR), capillary electrophoresis mass spectrometry (CE-MS), etc. What Bo Xiaoyuan wants to introduce to you below is a relatively new workflow, which was published in The Plant Journal in 2019 titled "Comprehensive mass spectrometry-guided phenotyping of plant specialized metabolites reveals metabolic diversity in the." Proposed in the cosmopolitan plant family Rhamnaceae" article, the article describes that the process is a scalable semi-automated method that digitally visualizes the diversity and distribution of plant metabolites by integrating several computational mass spectrometry/mass spectrometry data analysis methods.
Figure 3 Data analysis workflow for phenotypic analysis of plant-specific metabolites using tandem mass spectrometry (MS/MS) (Kang et al., 2019). Spectral similarity analysis is performed by tandem mass spectrometry, and similar spectra are clustered into subpopulations to form a molecular network. Network Annotation Propagation (NAP) provides silicon annotation candidates for a single spectrum. These candidate molecules are chemically classified using ClassyFire, and then the molecular families are hypothetically annotated according to the most dominant chemical categories in each molecular family. At the same time, the distribution of co-occurring fragments and neutral losses (Mass2Motifs) was analyzed by MS2LDA, which provided information on the diversity and distribution of substructures between samples.
Mass spectrometry imaging
Mass spectrometry imaging (MSI) is a mass spectrometry-based molecular ion imaging technique. Mass spectrometry can be obtained from regions of micron size on the surface, converting the lateral distribution of compounds on the surface (microelectronics, tissue sectioning) into an image, which in turn is associated with an optical image. Some common ionization techniques include DESI imaging, MALDI imaging, and secondary ion mass spectrometry imaging (SIMS imaging).
Capable of label-free detection and localization of a wide range of molecules on complex surfaces, MSI has become an attractive molecular histology tool in pharmaceutical and medical research. Since 2005, MSI has been gradually applied to plant research (Imai et al., 2005; Mullen et al., 2005). Spatial tissue information about proteins and metabolites will greatly improve our understanding of plant metabolism and the biochemical function of specific plant tissues (Lee et al., 2012; Matros and Mock, 2013).
The core of MSI experiments is the mass spectrometer, which consists of three parts: ion source, mass spectrometry analyzer and detector. In the ion source, the analytes are desorpted and ionized; in the analyzer, they are separated according to the mass-to-charge ratio (m/z); and finally, the separated ions are detected in the detector. As the final output, mass spectra are generated by displaying the intensity of the detected ions on the m/z scale. The basic principle of microprobe MSI is simple: the instrument collects a series of mass spectra by rasterizing specific regions of tissue samples. Subsequently, a map of the distribution of the analyte on the sample surface was generated by plotting the intensity of the analytes at individual m/z peaks and x-y coordinates in mass spectrometry (Goodwin et al., 2008; Svatos, 2010). The typical workflow of a MALDI imaging experiment is shown in Figure 4.
Figure 4 Typical MALDI imaging experiment flow.
Sample handling is the most critical step in MSI because only proper sample preparation can maintain the source, distribution, and abundance of molecules, ensuring high-quality signals and adequate spatial resolution (Grassl et al., 2011; Kaspar et al., 2011; Peukert et al., 2012; Spengler, 2014). MSI sample preparation methods for plant tissues are more challenging than for mammalian tissues (Boughton et al., 2015; Heyman and Dubery, 2016). The stratum corneum of higher plant bodies, which represents internal metabolites, is the first obstacle to direct mass spectrometry imaging because most softening techniques, such as MALDI and DESI, have difficulty penetrating them (Thunig et al., 2011). Plant cell walls are the second barrier for intracellular molecular mass spectrometry imaging. For example, when a matrix solution is sprayed onto the surface of a plant body, the cell wall prevents the solution from spreading over the cell wall, resulting in less efficient analyte extraction in MALDI imaging (Takahashi et al., 2015). The high moisture content in plant tissues presents another challenge during cryo-sectioning. Plant tissue is more fragile when frozen, and it is also more difficult to obtain intact thin sections for water-rich plant samples. Sample shrinkage or partial spalling is often observed during dehydration, a phenomenon that often leads to uneven surfaces that affect MSI analysis. It should be noted that the shrinkage of the sample can also lead to mismatches between THE MS images and the optical images (Cha et al., 2008), making biological interpretation difficult. Below are examples of the application of mass spectrometry imaging in plant research.
Figure 5 Mass spectrometry imaging of sub-stratum corneal metabolites. (i) MALDI imaging of Arabidopsis thaliana leaves. Clamp the left side of the leaf with forceps, leave it untreated in the middle, and dip the right side with chloroform (b). Kaempferol (a) and kaempferol rhamnoside (d) are easily detected in clamping and chloroform soaking areas, while fatty acids C26(c) and C30(f) are washed off in chloroform soaking areas. The unidentified analyte m/z =210(d) showed high abundance in the chloroform impregnated region. (ii) DESI image of hydroxybutyronitrile of barley leaf epidermis m/z = 276(b), 298(c), and 300(d). The dorsal epidermal band is peeled off (a). (iii) Blot imaging versus direct DESI imaging. (A) Indirect DESI imaging of leaf prints on PTFE. (B) Direct DESI imaging with chloroform, methanol and water (1:1:0.4, v/v/v) as spray solvents. (a) Optical image of the leaf blade of the centroid root. (a1) Optical image of leaf imprints on PTFE. (b-e) DESI images of m/z 104, 286, 298 and 300, respectively. These data were synthesized from (Cha et al., 2008), (Li et al., 2011), (Li et al., 2013) and (Bjarnholt et al., 2014).
Plant-based MSI previously focused on the methodological aspect (Matros and Mock, 2013), but now that the technology has matured, it has also been used to solve biologically important problems (Lee et al., 2012). For example, in plant environmental interactions (Shroff et al., 2008, 2015; Klein et al., 2015; Ryffel et al., 2015; Soares et al., 2015; Tata et al., 2015), new compound identification (Jaeger et al., 2013; Debois et al., 2014) and functional genomics (Korte et al., 2014) 2012;Li et al., 2013) has some applications. If you are interested, you can find out for yourself!
Summary
Through the introduction of the above aspects, Xiao Yuan believes that you should have a general understanding of mass spectrometry, for people who do not understand mass spectrometry at all, after reading today's article, there may still be a situation that seems to understand non-understanding, it does not matter if you do not understand, as long as you know what aspects of mass spectrometry are used, and when you do related experiments in the future, you can think of mass spectrometry, Xiao Yuan even if you did not write this article in vain! In any case, mass spectrometry is just an experimental tool, is to serve scientific research, as a scientific researcher you have to do is how to make good use of it, let it play a greater value!
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