The field of plant hormonomics focuses on the qualitative and quantitative analysis of the hormone complement in plant samples, akin to other omics sciences. Plant hormones, alongside primary and secondary metabolites, govern vital processes throughout a plant's lifecycle. While active hormones have received significant attention, studying all related compounds provides valuable insights into internal processes. Conventional single-class plant hormone analysis employs thorough sample purification, short analysis and triple quadrupole tandem mass spectrometry. Conversely, comprehensive hormonomics analysis necessitates minimal purification, robust and efficient separation and better-performing mass spectrometry instruments. This review summarizes the current status of plant hormone analysis methods, focusing on sample preparation, advances in chromatographic separation and mass spectrometric detection, including a discussion on internal standard selection and the potential of derivatization. Moreover, current approaches for assessing the spatiotemporal distribution are evaluated. The review touches on the legitimacy of the term plant hormonomics by exploring the current status of methods and outlining possible future trends.

• Keywords
• Hormonomics, Internal standard, Liquid chromatography, Mass spectrometry, Matrix effect, Metabolomics, Omics, Plant hormone, Solid phase extraction,
• Publication type
• Journal Article MeSH
• Review MeSH

Polar columns used in the HILIC (Hydrophilic Interaction Liquid Chromatography) systems take up water from the mixed aqueous-organic mobile phases in excess of the water concentration in the bulk mobile phase. The adsorbed water forms a diffuse layer, which becomes a part of the HILIC stationary phase and plays dominant role in the retention of polar compounds. It is difficult to fix the exact boundary between the diffuse stationary and the bulk mobile phase, hence determining the column hold-up volume is subject to errors. Adopting a convention that presumes that the volume of the adsorbed water can be understood as the column stationary phase volume enables unambiguous determination of the volumes of the stationary and of the mobile phases in the column, which is necessary for obtaining thermodynamically correct chromatographic data in HILIC systems. The volume of the aqueous stationary phase, Vex, can be determined experimentally by frontal analysis combined with Karl Fischer titration method, yielding isotherms of water adsorbed on polar columns, which allow direct prediction of the effects of the composition of aqueous-organic mobile phase on the retention in HILIC systems, and more accurate determination of phase volumes in columns and consistent retention data for any mobile phase composition. The n phase volume ratios of 18 columns calculated according to the new phase convention strongly depend on the type of the polar column. Zwitterionic and TSK gel amide and amine columns show especially strong water adsorption.

• Keywords
• hydrophilic interaction, liquid chromatography, polar columns, stationary and mobile phase,
• Publication type
• Journal Article MeSH

Glycoproteomics is a challenging branch of proteomics because of the micro- and macro-heterogeneity of protein glycosylation. Hydrophilic interaction liquid chromatography (HILIC) is an advantageous alternative to reversed-phase chromatography for intact glycopeptide separation prior to their identification by mass spectrometry. Nowadays, several HILIC columns differing in used chemistries are commercially available. However, there is a lack of comparative studies assessing their performance, and thus providing guidance for the selection of an adequate stationary phase for different glycoproteomics applications. Here, we compare three HILIC columns recently developed by Advanced Chromatography Technologies (ACE)- with unfunctionalized (HILIC-A), polyhydroxy functionalized (HILIC-N), and aminopropyl functionalized (HILIC-B) silica- with a C18 reversed-phase column in the separation of human immunoglobulin G glycopeptides. HILIC-A and HILIC-B exhibit mixed-mode separation combining hydrophilic and ion-exchange interactions for analyte retention. Expectably, reversed-phase mode successfully separated clusters of immunoglobulin G1 and immunoglobulin G2 glycopeptides, which differ in amino acid sequence, but was not able to adequately separate different glycoforms of the same peptide. All ACE HILIC columns showed higher separation power for different glycoforms, and we show that each column separates a different group of glycopeptides more effectively than the others. Moreover, HILIC-A and HILIC-N columns separated the isobaric A2G1F1 glycopeptides of immunoglobulin G, and thus showed the potential for the elucidation of the structure of isomeric glycoforms. Furthermore, the possible retention mechanism for the HILIC columns is discussed on the basis of the determined chromatographic parameters.

• Keywords
• Glycopeptides, Glycoproteomics, Hydrophilic interaction liquid chromatography, Immunoglobulin G, Mixed-mode chromatography,
• MeSH
• Chromatography, Ion Exchange methods   MeSH
• Chromatography, Reverse-Phase methods   MeSH
• Glycopeptides isolation & purification   MeSH
• Hydrophobic and Hydrophilic Interactions MeSH
• Immunoglobulin G isolation & purification   MeSH
• Isomerism MeSH
• Humans MeSH
• Proteomics MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Comparative Study MeSH
• Names of Substances
• Glycopeptides MeSH
• Immunoglobulin G MeSH

The composition of a sample solvent has a crucial impact on separations in hydrophilic interaction liquid chromatography (HILIC). In this short communication, we studied the effect of an organic modifier in the sample solvent on the solubility of different tryptic glycopeptides of hemopexin and haptoglobin proteins. The results showed that the solubility of glycopeptides in solvents with a high acetonitrile content depends on the type of attached N-glycan. We observed lower solubility in larger glycans attached to the same peptide backbone, and we demonstrated that glycopeptides containing sialic acids precipitate more readily than those without sialic acid. Therefore, the sample solvent composition in HILIC must be carefully optimized for accurate quantitative data collection and for adequate separation.

• Keywords
• Glycopeptides, Hydrophilic interaction liquid chromatography, Solubility,
• MeSH
• Acetonitriles chemistry   MeSH
• Glycopeptides analysis   chemistry   isolation & purification   MeSH
• Hydrophobic and Hydrophilic Interactions MeSH
• N-Acetylneuraminic Acid chemistry   MeSH
• Polysaccharides chemistry   MeSH
• Solvents chemistry   MeSH
• Chromatography, High Pressure Liquid methods   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• acetonitrile MeSH Browser
• Acetonitriles MeSH
• Glycopeptides MeSH
• N-Acetylneuraminic Acid MeSH
• Polysaccharides MeSH
• Solvents MeSH