An antibody-free enrichment approach enabled by reductive glutaraldehydation for monomethyllysine proteome analysis.

Protein lysine monomethylation is an important post-translational modification participated in regulating many biological processes. There is growing interest in identifying these methylation events. However, the introduction of one methyl group on lysine residues has negligible effect on changing the physical and chemical properties of proteins or peptides, making enriching and identifying monomethylated lysine (Kme1) proteins or peptides extraordinarily challenging. In this study, we proposed an antibody-free chemical proteomics approach to capture Kme1 peptides from complex protein digest. By exploiting reductive glutaraldehydation, 5-aldehyde-pentanyl modified Kme1 residues and piperidine modified primary amines were generated at the same time. The peptides with aldehyde modified Kme1 residues were then enriched by solid-phase hydrazide chemistry. This chemical proteomics approach was validated by using several synthetic peptides. It was demonstrated that it can enrich and detect Kme1 peptide from peptide mixture containing 5000-fold more bovine serum albumin tryptic digest. Besides, we extended our approach to profile Kme1 using heavy methyl stable isotope labeling by amino acids in cell culture (hmSILAC) labeled Jurkat T cells and Hela cells. Totally, 29 Kme1 sites on 25 proteins were identified with high confidence and 11 Kme1 sites were identified in both two types cells. This is the first antibody-free chemical proteomics approach to enrich Kme1 peptides from complex protein digest, and it provides a potential avenue for the analysis of methylome.

Global profiling of arginine dimethylation in regulating protein phase separation by a steric effect-based chemical-enrichment method.

Protein arginine methylation plays an important role in regulating protein functions in different cellular processes, and its dysregulation may lead to a variety of human diseases. Recently, arginine methylation was found to be involved in modulating protein liquid-liquid phase separation (LLPS), which drives the formation of different membraneless organelles (MLOs). Here, we developed a steric effect-based chemical-enrichment method (SECEM) coupled with liquid chromatography-tandem mass spectrometry to analyze arginine dimethylation (DMA) at the proteome level. We revealed by SECEM that, in mammalian cells, the DMA sites occurring in the RG/RGG motifs are preferentially enriched within the proteins identified in different MLOs, especially stress granules (SGs). Notably, global decrease of protein arginine methylation severely impairs the dynamic assembly and disassembly of SGs. By further profiling the dynamic change of DMA upon SG formation by SECEM, we identified that the most dramatic change of DMA occurs at multiple sites of RG/RGG-rich regions from several key SG-contained proteins, including G3BP1, FUS, hnRNPA1, and KHDRBS1. Moreover, both in vitro arginine methylation and mutation of the identified DMA sites significantly impair LLPS capability of the four different RG/RGG-rich regions. Overall, we provide a global profiling of the dynamic changes of protein DMA in the mammalian cells under different stress conditions by SECEM and reveal the important role of DMA in regulating protein LLPS and SG dynamics.

Fabrication of hydrophilic titanium (IV)-immobilized polydispersed microspheres via inverse suspension polymerization for enrichment of phosphopeptides in milk.

It is prerequisite to efficient extraction of phosphopeptides in bottom-up strategy for protein phosphorylation research. A kind of Ti4+-immobilized polydispersed phosphate-rich microsphere was fabricated via inverse suspension polymerization by employing vinylphosphonic acid (VPA) and N,N-methylenebisacrylamide (MBA) as functional monomer and crosslinker, respectively. The resulting microsphere demonstrated excellent sensitivity (as low as 10 fmol), selectivity (the mass ratio of ß-casein to BSA digests is 1/500), and adsorption capacity (up to 200 mg g-1). What's more, 113 unique phosphopeptides assigned to 25 unique phosphoproteins were indiscriminately identified from 5 µL of pasteurized milk digest, exhibiting great performance in capturing phosphopeptides. In this approach, only two steps were required to synthesize Ti4+-IMAC (Ti4+-Immobilized metal affinity chromatography). Compared with other methods required multistep modifying process, this strategy is simple and time-saving, offering a prospect of pilot production and commercialization. It is expected that the application of IMAC in milk and other food samples will still make it possible to unravel the huge complexity of the Foodome in the near future.

Proteome-Wide Deconvolution of Drug Targets and Binding Sites by Lysine Reactivity Profiling.

Recently, numerous efforts have been devoted to identifying drug targets and binding sites in complex proteomes, which is of great importance in modern drug discovery. In this study, we developed a robust lysine reactivity profiling method to systematically study drug-binding targets and binding sites at the proteome level. This method is based on the principle that binding of a drug to a specific region of target proteins will change the reactivity of lysine residues that are located at this region, and these changes can be detected with an enrichable and lysine reactive probe. Coupled with data-independent acquisition (DIA), the known target proteins and corresponding binding sites were successfully revealed from K562 cell lysates for three model drugs: geldanamycin, staurosporine, and dasatinib. In addition, the drug-induced conformational changes of certain targets were also revealed by our method during the screening of staurosporine. The screening sensitivity of our method revealed from the screening of stuarosporine and dasatinib was comparable with that of thermal proteome profiling (TPP) or machine learning-based limited proteolysis (LiP-Quant). Overall, 21 and 4 kinase targets, including adenosine 5'-triphosphate (ATP)-binding targets, were identified for staurosporine and dasatinib in K562 cell lysates, respectively. We found that target proteins identified by TPP, LiP-Quant, and our method were complementary, emphasizing that the development of new methods that probe different properties of proteins is of great importance in drug target deconvolution. We also envision further applications of our method in proteome-wide probing multiple events that involve lysine reactivity changes.

An efficient approach based on basic strong cation exchange chromatography for enriching methylated peptides with high specificity for methylproteomics analysis.

Protein methylation as one of the most important post-translational modifications has been under the spotlight due to its essential role in many biological processes. Development of methods for large-scale analysis of protein methylation greatly accelerates the related researches. To date, antibody-based enrichment strategy is the most common approach for methylproteomics analysis. However, it is still lacking of a pan-specific antibody to enrich peptides or proteins carrying all kinds of lysine and arginine methylation forms. Herein, an online basic strong cation exchange chromatography was developed to enrich methylated peptides from protein digests prepared by two complementary methods, including direct multiple enzymes digestion and carboxylic amidation followed by multiple enzymes digestion. After enrichment, the majority of identifications were obtained from direct multiple enzymes digested sample. The enrichment specificity of methylated peptides was up to 28.5%, and 445 methylation forms corresponding to 376 methylation sites were identified on 194 proteins in one LC-MS/MS run using only 100 µg of digests. This method has great potential in studying protein methylation mediated biological processes.

Chemical Depletion of Histidine-Containing Peptides Allows Identification of More Low-Abundance Methylation Sites from Proteome Samples.

Protein methylation, especially that occurs on arginine and lysine residues, is one of the most important post-translational modifications involved in various cellular processes including RNA splicing, DNA repair, and so forth. Systematic analysis of protein methylation would facilitate the understanding of its regulatory mechanisms. Strong cation chromatography has been used to globally analyze arginine/lysine methylation at the proteome scale with good performance. However, the co-enriched histidine-containing peptides severely interfere with the detection of low-abundance methylpeptides. Here, we developed a novel chemical strategy which enabled almost complete depletion of histidine-containing peptides in the protein digest, thereby resulting in the identification of more low-abundance arginine/lysine methylpeptides. Totally, 333 arginine and lysine methylation forms from 207 proteins were identified in this study. Overall, the number of methylation identifications increased about 50% by using our new method. Data are available via ProteomeXchange with the identifier PXD023845.

Selective enrichment of N-terminal proline peptides via hydrazide chemistry for proteomics analysis.

A challenge for shotgun proteomics is the identification of low abundance proteins, which is always hampered owing to the extreme complexity of protein digests and highly dynamic concentration range of proteins. To reduce the complexity of the peptide mixture, we developed a novel method to selectively enrich N-terminal proline peptides via hydrazide chemistry. This method consisted of ortho-phthalaldehyde (OPA) blocking of primary amines in peptides, reductive glutaraldehydation of N-terminal proline and solid phase hydrazide chemistry enrichment of aldehyde-modified N-terminal proline peptide. After enrichment, the number of detected peptides containing N-terminal proline increased from 1304 to 4039 and the ratio of N-terminal proline peptides jumped from 4.4% to 93.7%, showing good enrichment specificity towards N-terminal proline peptides. Besides, the ratio of identified peptides to proteins was decreased from 7.8 (29751/3811) to 1.5 (4347/2821), indicating that sample complexity was drastically reduced through this method. As a result, this novel approach for enriching N-terminal proline peptides is effective in identification of low abundance protein owing to the reduction of sample complexity.

In situ growth of COF-rLZU1 on the surface of silica sphere as stationary phase for high performance liquid chromatography.

The composite materials consist of Covalent Organic Frameworks (COFs) and silica have been regarded as a kind of promising stationary phases due to combination of the large specific surface area and good mechanical strength of porous silica microspheres and the porous structure and the excellent stability of COFs. Herein, a novel COFs-silica composite (SiO2@rLZU1, reduced Lan Zhou University-1) was prepared via an in-situ growth strategy with a 32 nm-thick COFs layer on the surface of silica and a 2.2 nm-thick COFs layer on the inner surface of the mesopores of spherical silica (5 µm, 120 Å). With secondary amine and phenyl groups, the novel stationary phase provided hydrophilic, hydrophobic and π-π interactions when used in HPLC, showing different selectivity from typical reversed-phase stationary phases. Probe molecules with aromatic moieties varying in polarity, including acidic (phenol, pyrocatechol, and pyrogallol), basic (aniline, 4-chloroaniline, and 4-nitroaniline) and neutral (benzene homologues) compounds, were all baseline separated on the SiO2@rLZU1 column, indicating its excellent separation performance. Besides, the SiO2@rLZU1 column also exhibited great repeatability with intraday RSDs of the retention time of three anilines less than 0.31% (n = 6) and peak area less than 1.63% (n = 6). On the SiO2@rLZU1 column, satisfied results were achieved in the separation of real samples such as fullerenes and coking wastewater, suggesting the great potential of the as-synthesized stationary phase in HPLC applications.