Common Post-translational Modifications (PTMs) of Proteins: Analysis by Up-to-Date Analytical Techniques with an Emphasis on Barley

Post-translational modifications (PTMs) of biomacromolecules can be useful for understanding the processes by which a relatively small number of individual genes in a particular genome can generate enormous biological complexity in different organisms. The proteomes of barley and the brewing process were investigated by different techniques. However, their diverse and complex PTMs remain understudied. As standard analytical approaches have limitations, innovative analytical approaches need to be developed and applied in PTM studies. To make further progress in this field, it is necessary to specify the sites of modification, as well as to characterize individual isoforms with increased selectivity and sensitivity. This review summarizes advances in the PTM analysis of barley proteins, particularly those involving mass spectrometric detection. Our focus is on monitoring phosphorylation, glycation, and glycosylation, which critically influence functional behavior in metabolism and regulation in organisms.


INTRODUCTION
The introduction of a new structural element into the protein molecule after translation of the mRNA code allows the polypeptide chain to be transformed into different species with different functions.All living organisms must respond rapidly and efficiently to environmental changes through a tight regulatory system via molecular interactions of several hundred biomolecules. 1Due to multiple levels of regulation, such as PTMs and alternative splicing, a single gene is capable of producing several different proteins. 2lternative uses of start and stop codons can give rise to different proteins.Proteins synthesized from these mRNAs may be differentially modified during or after translation.Hence, the same protein can be altered in many ways, leading to different variants.Since a cell is not a static entity, it is constantly responding to stimuli from both the external and internal environment. 3Therefore, many proteins are modified during or after synthesis in several ways, e.g., by cleavage of the polypeptide skeleton or by chemical modification of specific amino acid side chains.Post-translational modifications (PTMs serve a variety of purposes in different cellular processes, such as regulating enzymes, transducing signals, mediating subcellular localization of proteins, and interacting with proteins and different molecules. 4,5he PTMs can significantly affect protein localization and function.The identification and characterization of PTMs is therefore essential for understanding the regulation of metabolism, gene expression, and signaling in cells, and thus for the development of new therapeutics, as well as for the cultivation of high-quality plant varieties.

PTMs
Post-translational modifications can be categorized as follows: covalent incorporation of a chemical function to the side chain of a residue 6 and cleavage of the peptide backbone. 7In the former case, a given protein may be post-translationally modified at many residues by the same group or the protein may undergo the addition of multiple types of covalently introduced groups.More than 400 different types of PTMs have been described to date. 8However, only some of these have been characterized in detail at the proteome level.
A wide range of different approaches has been used to study PTMs.A number of these approaches target individual proteins; for example, protein-specific phosphoantibodies are used to study cell signaling processes.More complex PTM analyses often depend on purification steps based on specific enrichment of modified proteins/peptides. 9.1.Types of Protein PTMs.The most studied protein PTMs include glycosylation, phosphorylation, ubiquitination, acylation, methylation, nitration, and acetylation. 10The list of different types of PTMs is shown in Table 1.Some modifications, such as glycosylation, are usually permanent.Also, proteolytic cleavage represents the most prevalent irreversible PTM that all proteins undergo during their life cycle.In contrast, phosphorylation is reversible, can be used to regulate the activity of proteins in response to intracellular and extracellular signals, and is frequently involved in signaling pathways.
2.1.1.Glycosylation.There are two major types of glycosylation: N-and O-linked.The former involves glycans attached via amide nitrogen to asparagine. 11The latter reaction takes place at the hydroxy group of serine and threonine residues.Some glycoproteins contain sialic acid residues, whose ionizable nature significantly alters the behavior of the oligosaccharide moiety.It is well-known that glycosidic linkages in glycopeptides are more labile than peptide linkages and fragmentation of the glycan moiety predominates. 12Glycopeptides generate glycan-specific oxonium ions such as at m/z 204 (HexNAc), 163 (Hex), 292 (NeuNAc), and 366 (Hex-HexNAc) useful for structure assignment. 13The ion at m/z 204, as well as ions at m/z 186 and 168 arising from the elimination of water molecules, has been shown to be indicative for both N-glycans and O-glycans. 14Also, mass spectra data sets can be validated for constant neutral loss of terminal monosaccharides. 15e analysis of PTMs of proteins mainly requires their selective enrichment due to the low stoichiometry of modifications relative to nonmodified proteins, low abundance, heterogeneity of derivatives, and the majority of interfering nonmodified proteins.For enrichment of glycopeptides, methods based on lectin chromatography, 16 hydrophilic interaction chromatography (HILIC), 17 reverse phase chromatography, electrostatic repulsion hydrophilic interaction chromatography (ERLIC), 18 cation-exchange chromatography, 19 boronic acid-functionalized particles, 20 and hydrazine chemistry were used. 21Affinity chromatography with lectins shows the changes in glycosylation patterns; 16 it possesses a high specificity for the particular type of glycan moiety.
Cellulose, amide, and zwitterionic phases were used for HILIC separation.It can enrich hydrophilic non-glycosylated peptides as impurities. 22Under reversed-phase LC conditions, glycopeptides have been enriched using porous graphite. 19lycopeptide resolution in ERLIC is based on the structure of glycans; it exhibits potential use for separating isomers caused by carbohydrate microheterogeneity.The principle predeterminate use of this method is for the enrichment of sialoglycopeptides. 18lycopeptides containing sialic acid can be retained very efficiently on TiO 2 under conditions in which unmodified peptides or neutral glycopeptides do not bind. 23Contamination with phosphorylated peptides is suppressed by phosphatase treatment, whereas the presence of sulfated peptides is not ruled out. 24Boronate affinity chromatography (BAC) is characterized by formation of covalent bonds between immobilized boronic acid and a cis arrangement of vicinal hydroxy groups.Gold nanoparticles functionalized with phenylboronic acid were utilized for the on-plate specific selective concentration of glycosylated peptides before matrix-assisted laser desorption/ ionization (MALDI) analysis.However, BAC is unique not only for glycopeptides but for all compounds containing cis-diol moieties. 25he extraction selectivity can be improved by the conjunction of independent separation principles.Zwitterionic HILIC and RP-18 chromatography is orthogonal and complementary for the separation of glycopeptides.While HILIC distinguishes the glycan group, RP-18 facilitates separation by peptide sequence and degree of sialylation.
2.1.1.1.Formation of N-Glycoproteins.In plants, the most widely studied protein modification is N-glycosylation.The oligosaccharide precursor Glc3Man9GlcNAc2 (glucose-man- The number of published papers was obtained by entering the modification type in the Web of Science (October 2022).nose-N-acetyl-D-glucosamine) first attaches to the Asn residue, and the formed glycoprotein is then moved from the ER via the Golgi apparatus to its destination.During this process, N-glycan undergoes several changes consisting of the final removal and addition of sugar groups by different glycosidases and glycosyltransferases. 26 A potential N-X-S/T glycoprotein sequence may be present multiple times in a polypeptide chain, but not all potential N-glycosylation sites are necessarily occupied. 27lant N-glycans differ from their animal counterparts in several properties. 28As an example, plants do not produce and activate sialic acid and are unable to perform β(1,4)galactosylation.Several attempts have been made to achieve this.The formation of stable protein sialylation in Physcomitrella has been described by Bohlender et al. 29 2.1.2.Nonenzymatic Glycosylation (Glycation).Proteins and peptides are very often modified by nonenzymatic glycation.This reaction is also called the Maillard reaction, whose chemistry is highlighted in Figure 1. 30 The free amine groups of lysine and guanidine groups of arginine in peptides and proteins react with reducing carbohydrates, α-oxoaldehydes, and their derivatives to form Schiff bases, which are not stable and are rapidly rearranged to the so-called Amadori products.
2.1.3.Phosphorylation.Phosphorylation usually occurs on serine, threonine, and tyrosine residues.Phosphorylation is reversible and controlled by the action of kinases and phosphatases.Another important acidic moiety introduced during PTM is the SO 3 H group, which is formed by oxidation of the sulfanyl group of cysteine or transferred to hydroxyls of tyrosine, serine, or threonine in proteins. 31dentification of phosphopeptides is deduced from the neutral loss of 98 (Ser, Thr) or 80 (Ser, Thr, Tyr) Da in positive-ion mode and, respectively, 79 or 63 (Ser, Thr, Tyr) in negative-ion mode.This procedure, combined with the phosphatase treatment, leads to a mass shift of 80 Da. 32 Unlike the phosphorylated peptides, which retained the moiety at lowenergy collision, isobaric sulfated peptides lost SO 3 (80 Da) before backbone fragmentation occurred. 33Moreover, the instruments can be capable of measuring weight with high accuracy to distinguish phosphate versus a sulfate group based on their mass difference (9.5 mDa). 34solation of phosphopeptides involves immunoprecipitation with phosphospecific antibodies, 35,36 immobilized metal affinity chromatography (IMAC), 32 metal oxide affinity chromatography (MOAC), HILIC, ERLIC, and strong anion exchange chromatography (SAX). 37 technique based on cation exchange separation has been developed for the enrichment of Tyr sulfated peptides.Sulfation sites are characterized by the presence of several acidic amino acids in their vicinity. 38For the detection, purification, and monitoring of sulfonation of tyrosine-sulfated proteins, monoclonal antibodies have been produced. 39everal described isolation approaches show broad specificity, and therefore, they can potentially simultaneously enrich various post-translationally modified peptides.Multistep parallel screening of glycosylation and phosphorylation was suggested by Zhao et al. 40 Some results demonstrating simultaneous purification of neutral glycopeptides and phosphopeptides on porous titania microspheres have been published.

PROTEIN PTMs AND INNOVATIVE ANALYTICAL TECHNIQUES
Traditionally, PTMs have been identified by Edman degradation, 41 radioactive isotope labeling, 42 and Western blot immunoanalysis. 43sing Edman cleavage, the amino acid sequence of a given protein or peptide can be determined.The method allows the peptide to be labeled and cleaved from the N-terminus without breaking the peptide bonds between the other amino acid residues.Unfortunately, this method is not suitable for the analysis of shorter sequences. 44In 2020, Kristoffersen used Edman degradation sequencing and immunological detection to separate and characterize essential barley seed proteins. 45Their work identified three hordeins and detected a fourth hordein, while other partially sequenced proteins appear to have roles in plant stress or defense.
Due to its high sensitivity, mass spectrometry is a versatile tool for locating and mapping PTM sites.MS-based strategies typically involve several steps: enzymatic digestion of the protein, concentration of modified peptides, peptide sequencing by MS/MS, and statistical evaluation of results using databases.MALDI, electrospray ionization (ESI) mass spectrometry, and surface-enhanced laser desorption/ionization MS are frequently employed for this purpose. 46The shotgun sequencing approach is based on the parallel action of multiple proteases with different specificities with analysis by the HPLC-MS/MS technique.It is useful to convert PTMs that are labile during MS(/MS) experiments to stable species, preferably by introduced tagging. 47ollision induced dissociation (CID) and electron capture/ transfer dissociation (ECD/ETD) usually give complementary information on glycopeptides.In CID, fragmentation of protonated glycopeptides occurs mainly at glycosidic linkages, producing carbohydrate oxonium ions and/or fragment ions resulting from the repeated loss of monosaccharide units from both ends of the glycan. 48The schematic representation of proteomic MS-based strategies commonly used for PTM analysis is depicted in Figure 2.
It is well-known that an appropriate matrix selection for MS measurements often determines the outcome.The binary matrices 2,5-dihydroxybenzoic acid (2,5-DHB)/α-cyano-4hydroxycinnamic acid (CHCA) and 2,5-DHB/sinapinic acid (SA) can also be used for MALDI-MS analysis of glycoproteins and determination of other modified proteins. 49apillary electrophoresis (CE) also offers a simple application for glycan analysis.Since most glycans and oligosaccharides generally do not contain a chromophore or fluorophore in their natural structure and are difficult to ionize, it is necessary to perform a labeling step prior to their analysis using MS.Recently, several new labels and labeling strategies have emerged for carbohydrate analysis using CE.Labeling of carbohydrate molecules with a carbonyl group can be achieved by several reactions.It should be mentioned that the most common chemical labeling method is the reductive amination reaction, which is used to derivatize carbohydrates.At present, labeling with 8-aminopyrene-1,3,6-trisulfonic acid trisodium salt (APTS) is mainly used. 50lassical approaches have limitations such as a gap between shotgun-based approaches and clinically relevant results.Therefore, new approaches, sample preparations, and instrument innovations need to be introduced.This includes the involvement of, e.g., microchip electromigration methods, advanced electromigration methods with MS detection, and a combination of nanotechnology, surface enzyme reactions, and microfluidic networks. 51,52he use of microfluidics in analyzing glycoproteins and glycans has become prevalent.One of the many systems researchers choose is the 1200 Infinity Series HPLC-Chip/MS instrument, which features a ChipCube interface for ESI.The columns, injection channel, ESI tip, and connections are housed on a laminated polyimide chip, which is inserted into the ChipCube interface in a credit-card-sized stainless-steel holder, with the Infinity LC instrument serving as the solvent delivery system.In 2019 Novotnýet al. 53 introduced a microfluidic device for isolation and purification of glycoprotein samples.As seen in Figure 3, the microfluidic chip contained a bed of microbeads coupled to immobilized biomolecules that served as a bioaffinity column.The dosing of the different fluids required for each step of the sample treatment procedure was controlled by integrated pneumatic valves.The microfluidic device was combined with the label-free voltammetric analysis using catalytic peak H, which also represents an alternative method for protein detection.The advantage of using peak H is that all proteins mainly produce this intrinsic signal.
Combinatorial peptide ligand libraries (CPLLs) designed for low-abundance proteins are also used in proteomics.Eliminating high-abundance proteins is very efficient, but detecting lowabundance proteins remains limited due to their dilute presence.What seems interesting about reducing the number of high abundance proteins while increasing the number of low abundance proteins is the possibility of detecting more proteins and their PTMs. 54CPLL technology is characterized by three important principles: (1) a mixed set of affinity resins covering the largest spectrum of affinity ligands to statistically provide a partner ligand for each protein present in the samples, (2) the saturation effect of the capture due to a large number of sample overload, and (3) the exhaustiveness of protein recovery for full analysis.Although CPLL technology is currently used to track low abundance proteins, some research groups are still comparing the immunodepletion method with enrichment methods to obtain the best results. 54ata-dependent acquisition (DDA) and data-independent acquisition (DIA) methods along with sequential window acquisition of all theoretical mass spectra (SWATH-MS) are also more widely used for protein identification and quantification. 55TRAQ, an advanced MS-based procedure, is also used for the relative and absolute quantification of proteins and their modification by the derivatization of primary NH 2 groups in intact proteins.All peptides in a sample are labeled with an isobaric tag (a reporter group), individual samples with different ones.During the MS/MS experiments, the reporter groups produce different ions for a particular example (e.g., m/z 114 and 117).The ratios of intensities of the different reporter ions provide the relative abundances of protein in compared samples. 56iTRAQ is mainly used to analyze different cellular systems and to describe changes in protein expression related to cancer; it is also applied in other areas of agricultural research. 57,58o identify changes in the electrophoretic mobility of phosphoproteins, Okawara et al. developed the Phos-tag SDS-PAGE method.However, there was a problem with this technique; when SDS-PAGE and Phos-tag SDS-PAGE were used separately, detecting mobility shifts using Mn 2+ Phos-tag was often challenging.The authors solved this problem by diagonal electrophoresis with Phos-tag, in which SDS-PAGE and Phos-tag SDS-PAGE patterns are presented on a single gel. 59norganic nanofibrous materials based on titanium dioxide or zirconia have been developed for the selective and efficient enrichment of phosphopeptides for MALDI/MS detection.Compared to conventional materials, the present nanofibrous materials exhibit much higher permeability, allowing their use in column packing and pipet tip format without the need for high pressure. 60

POST-TRANSLATIONAL MODIFICATIONS OF BARLEY PROTEINS AND DIFFERENT APPROACHES TO THEIR DETERMINATION
Barley (Hordeum vulgare) is considered to be one of the first cereals grown for human consumption.It is known to have been the food of gladiators and soldiers.However, barley is grown worldwide for a number of reasons, mainly as a feed grain and also for malting purposes.Barley is used in the production of beer and whisky and is also used in small quantities as a coffee substitute.Two types of botanical barley are distinguished: tworow and six-row genotypes.Six-row barley generally has a higher protein concentration in the grain than two-row barley, but both types are suitable for malting after modification of the cultivation method.Two-row malting barley is traditionally grown in Europe, Australia, and South America, while six-row malting barley is more frequently grown in North America. 61arley grain consists of a mixture of carbohydrates, proteins, lipids, minerals, and other important substances. 62,63Although protein accounts for approximately 10% of dry matter; it has been studied in detail.Their quantity and composition influence the suitability and quality of the barley grain for the final use, with the finished beer containing approximately one-third barley protein. 64Many proteins are modified during the malting process.In this review, we selected the most crucial barley proteins, their chosen modification, and analytical strategies used for their identification over the past few years (Table 2).
4.1.Glycosylation.−70 Several published results confirmed that a combination of multiple omics approaches could be beneficial in identifying potential candidate genes, their pathways, and the covalent modification of proteins.Generally, lectin affinity chromatography in various combinations with gel electrophoresis, liquid chromatography (LC), and MS techniques is the most widespread approach for glycoprotein analysis. 65,66In quantitative proteomics, the iTRAQ method showed differential expression of selected barley allergenic proteins (e.g., pUP38, barwin) during food processing. 57he methods used in proteomics are also applicable to glycoproteomics.Nevertheless, the natural heterogeneity and

Glycation.
Glycation is the second most common modification of barley proteins (apart from proteolysis or protein degradation).Approximately 3 to 7% of the glycated proteins in beer may be responsible for the formation of haze and approximately 25% for the stability of foam.Among the components responsible for the formation of haze is a prolinerich peptide derived from hordeins ranging in size from 15 to 32 kDa.The carbohydrate components in this case consist mainly of hexose.2][73][74][75][76][77][78][79][80][81]69 Malt varieties differed in the extent of glycation of LTP1 and protein Z fragments. Thiwas reflected by ladders of MS peaks differing in mass by approximately 162 Da.Bobaĺováet al. 81 showed that some protein glycation occurred on the second day of malting.An efficient method for rapid identification of malt proteins and determination of PTMs, particularly glycation, is the application of monolithic chromatographic media and analysis of intact proteins by MALDI-TOF MS.Fractionation of aqueous barley extract on monolithic convective interaction media (CIM) disks with diethylamine (DEAE) column yielded better MS spectra and allowed rapid detection of technologically induced glycation of the protein fragment Z (m/z 4.0 kDa), LTP1 (m/z 7.5 kDa) and LTP2.79 As shown in Table 2, previous studies have characterized glycated proteins by gel electrophoresis and mass spectrometry.Some authors have also used a combination of LC and mass spectrometry to examine protein structure.73,76 Cho et al. 82 used 2D-LC-MS/MS to enrich and analyze glycated proteins.Also, linear mode MALDI-MS can be used to monitor the malting process by characterizing the glycation of LTP proteins and protein Z, which is a fast and inexpensive analysis.78 Isoelectric focusing (IEF) devices appear to be an interesting alternative to LC.Their advantages are that they are significantly more straightforward, are cheaper than LC, and are faster than PAGE. Thmethod can also be used to study PTMs without special chemical requirements, sample preparation, and purification procedures.Mazanec et al. applied this approach for the study of nonenzymatic glycosylation of major barley proteins LTP and protein Z. 77 4.3.Phosphorylation.Most of the structural and metabolic proteins summarized in Table 2 belong to the phosphoprotein family.Within the previously mentioned mass spectrometrybased approaches, a wide range of enrichment techniques is used to capture phosphoproteins/phosphopeptides.[83][84][85][86][87]67 Recently, a phosphoproteomic study of barley grain based on IMAC or TiO 2 affinity chromatography followed by LC-MS/MS analysis was used to compare phosphoprotein regulation during imbibition.Phosphosignaling networks in barley grains were examined using large-scale phosphopeptide analysis to explore potential changes in ripening response pathways.83 Generally, study of phosphoprotein profiles indicates the major influence of protein phosphorylation in seed germination and can serve for monitoring of physiological changes occurring during germination and dormancy, as well as stress conditions.84,85 In recent years, immunoaffinity strategies have been widely used for the enrichment of phosphoproteins involved in various biological processes and metabolic pathways, such as Ca 2+ signaling pathways, carbohydrate metabolism, or signaling pathways.87,88 In addition, Wang et al. 87 performed a coimmunoprecipitation (co-IP) assay (using phospho-serine) and MS/MS analysis for specific KEGG pathway enrichment.The results show that phosphoproteins, found mainly in the nucleus and chloroplast, play a key role in processes of the MAPK signaling pathway such as RNA transport, endocytosis, or RNA degradation.
Finally, a brief overview of the essential proteins and their PTMs identified in barley is given in Table 3.We focused on the three most important groups of proteins classified according to their biological functions: (a) storage proteins (prolamins/ hordeins); (b) structural and metabolic proteins; and (c) pathogenesis-related proteins.Structural and metabolic proteins, mainly kinases, transferases, and glycoside hydrolases, represent the main proteins studied in this review.As expected, protein glycosylation occurs predominantly in all three types of proteins: storage proteins, pathogenesis-related proteins, and structural and metabolic proteins.According to the database, some of the identified proteins have also been described as significant sensitizers.It is also known that N-linked glycans of plant glycoproteins are among the most abundant environmental immune determinants.In contrast, nonenzymatic glycosylation is most common in pathogenesis-related proteins, particularly α-amylase inhibitors, serpins, and LTP proteins.
4.4.Alternative Modifications.Thanks to rapid advances in high-throughput mass spectrometry, it is now possible to comprehensively detect and quantify PTM sites within a single proteomics experiment, including phosphorylation and glycosylation, as well as ubiquitination, SUMOylation, acetylation, and succinylation.Succinylation, a type of PTM occurring on lysine residues, has been shown to play an important role in gene transcription and plant growth.Wang et al. recently suggested that protein phosphorylation and succinylation are often occurring PTMs that may play a key regulatory role in the response of barley roots to phosphate stress.This finding is interesting given that phosphate stress represents an important environmental factor limiting plant growth and development. 89ecause the function of small RHO-type G-proteins as signaling centers and key regulators of cell polarity must be strictly controlled, PTMs of RHO proteins, such as lipid modifications, phosphorylation, and ubiquitination, have recently been described.It was found that the ubiquitination site is maintained in all barley ROPs.This indicates that the lysine residue belonging to RACB K167 is a general target for ubiquitination and that this regulates the amount of protein. 90UMOylation is a key player in plant immunity and, thus, plays a positive role in fungal resistance.By studying the molecules accumulated in barley, a certain level of resistance to Fusarium graminearum was confirmed, leading to the identification of resveratrol, a candidate inhibitor of SUMO proteases. 91ur brief review concerning the PTMs of barley was mainly focused on the study of individual PTMs.Since studies of the interactions of PTMs in plants have only recently emerged, it will undoubtedly be desirable to focus attention on this area in the future.

OTHER IMPORTANT CROPS
As mentioned above, several efficient methods have been developed for N-glycoprotein enrichment according to different enrichment mechanisms.These methods were applied to the glycoproteomic analysis of various plant species, including Arabidopsis leaf cell walls, Brachypodium distachyon seedling leaves, ripe tomato fruits, maize seeds, and seedling leaves and aleurone layers of barley, wheat, and rice.Since wheat (Triticum aestivum L.) is a globally important cereal crop and PTMs of proteins are widely involved in the regulation of plant abiotic stress, Wang et al. performed the first analysis of the Nglycoproteome of wheat seedling leaves by enrichment glycosylation using the HILIC method and tandem mass spectrometric analysis with the use of an Orbitrap Q Exactive Plus hybrid quadrupole-Orbitrap mass spectrometer.The results of their study showed that glycosylation sites throughout the cell are more likely to be located on a random coil, and the study also showed that glycosylation modification maintains the stability of the protein structure. 92Because drought has a severe limiting effect on wheat growth and yield formation, a combination of a proteomics approach and Pro-Q Diamond gel staining was used to determine that several proteins are phosphorylated and up-regulated under drought conditions.Luo et al. identified 58 wheat proteins that were phosphorylated among 112 differentially accumulated proteins in response to the water deficit.Significantly, phosphorylation of heat shock proteins, for example, was specifically induced by drought stress, which may suggest an important role in drought resistance. 93ice (Oryza sativa L.) is also one of the most frequently consumed cereals.Recently, proteomic technology has been used to determine differentially expressed rice proteins related to starch biosynthesis and to identify PTMs that target starch biosynthesis proteins.Most proteins related to starch biosynthesis are essentially elevated at 6−20 days after flowering and decrease under conditions of high temperature.Some of these have been identified as being targeted by phosphorylation, lysine acetylation, succinylation, 2-hydroxyisobutyrylation of lysine, and malonylation.Phosphoglucomutase, an important enzyme that transfers the phosphate group on the α-D-glucose monomer from position 1 to position 6, is commonly the target of five types of PTMs. 94In the endosperm of maize (Zea mays L.), starch synthesis (SSI, SSIIa) and starch branching (SBEIIb) enzymes form a trimeric complex, with SBEIIb being phosphorylated. 95The formation of the complex is triggered by ATP and degrades alkaline phosphatase.These findings confirm the hypothesis that phosphorylation-dependent heteromeric enzyme complex formation facilitates amylopectin formation in starch granules. 96nother important task for cereals is the detection of allergenic proteins.Allergen characterization has recently been addressed by allergenomics, which can be used to identify putative allergens over a short time.Allergens present in cereal grains involve proteins belonging to the groups of albumins, globulins, prolamins, and glutenins. 97Among the important allergens in wheat and barley flour is the α-amylase/trypsin inhibitor group, most of which are glycated or glycosylated. 98arley LTP1 and protein Z4 have also been confirmed to be significant beer allergens. 99

CONCLUSION
The works discussed in this review report different techniques used to identify barley PTMs.Advances in sample preparation, mass spectrometry, and bioinformatics are enabling more accurate and faster identification, quantification, and characterization of proteins as well as their modifications.
Proteomic methods based on LC-MS/MS are currently available for the detection of different proteins and their PTMs due to their accuracy, precision, and sensitivity and robust quantitative capability.One of the most recent approaches uses Data Independent Acquisition (DIA)/Sequential Window Acquisition of All Theoretical Mass Spectra (SWATH) LC-MS/MS with bioinformatics workflows to identify and measure PTMs in order to investigate the underlying protein biochemistry of various samples.Extensive complexity and diversity of PTMs were found in the proteomes, particularly proteolysis from barley proteases, O-glycosylation of secreted yeast glycoproteins, and the glycation of barley proteins with malto-oligosaccharides.
Another method is the electrochemical analysis of glycoprotein samples by combining a microfluidic device with voltammetric analysis on amalgam electrodes.Glycoproteins are electroactive on electrodes containing carbon and mercury.Their intrinsic signals obtained by square wave voltammetry or chronopotentiometric constant current sampling are very high with an effective baseline correction.At the same time, they are more developed, and much lower protein concentrations can be measured compared to linear sweep voltammetry.The literature shows that the combination of electrochemical methods with microfluidic devices is sporadically used.However, it should be mentioned that electrochemical methods offer accurate, simple, and inexpensive analysis.
As all knowledge of the proteins involved, their biological role, structure, and function in food raw materials and final food products is essential for food processing, new low-cost, efficient, and robust analytical methods with higher sensitivity need to be developed.

Figure 2 .
Figure 2. Scheme of MS-based proteomic strategies for PTM analysis.

Figure 3 .
Figure 3. (A) Assembled microfluidic device.(B) UltraLink microbeads packed into the device.(C) The device is connected to a constantly pressurized fluid dispenser.Which fluid is injected is determined by the valves and CDA-manifold.Reprinted with permission from ref 53.Copyright 2019 John Wiley and Sons.

Table 1 .
List of Critical Post-translational Modifications a

Table 2 .
68,71iew of the Essential Glycosylated, Glycated, and Phosphorylated Barley Proteins and Used Analytical Approaches for Their Study nature of glycosylation make it difficult to identify and quantify.For this reason, specialized software is needed to identify the glycan component attached to peptides or proteins.The obtained MS/MS spectra of these species are more complex, because of the fragmentation of the glycans.In general, glycosylated species are usually less abundant than peptides due to glycan heterogeneity and reduced ionization efficiency during MS.Both labeled and unlabeled approaches are widely used to quantify glycosylation by MS, including DDA, DIA, and multiple reaction monitoring.11,71Kerretal.68recentlyusedDIA/SWATH-MS and reported very high PTM diversity in 23 commercial beers, focusing mainly on proteolysis and glycosylation.As shown in Table2, the main group of highmannose N-glycosylated proteins was represented by some wellknown barley-pathogenesis-related proteins, such as protease/ α-amylase inhibitors, germin-like proteins, peroxidase, and hordeins. dynamic

Table 3 .
Summary of Essential Proteins and their PTMs Identified in Barley