Diagnostic Potential of Serum Glycome Analysis in Lung Cancer: A Glycopattern Study.

Lung cancer is the leading cause of cancer-related death, with high morbidity and mortality rates due to the lack of reliable methods for diagnosing lung cancer at an early stage. Low-dose computed tomography can help detect abnormal areas in the lungs, but only 16% of cases are diagnosed early. Tests for lung cancer markers are often employed to determine genetic expression or mutations in lung carcinogenesis. Serum glycome analysis is a promising new method for early lung cancer diagnosis as glycopatterns exhibit significant differences in lung cancer patients. In this study, we employed a solid-phase chemoenzymatic method to systematically compare glycopatterns in benign cases, adenocarcinoma before and after surgery, and advanced stages of adenocarcinoma. Our findings indicate that serum high-mannose levels are elevated in both benign cases and adenocarcinoma, while complex N-glycans, including fucose and 2,6-linked sialic acid, are downregulated in the serum. Subsequently, we developed an algorithm that utilizes 16 altered N-glycans, 7 upregulated and 9 downregulated, to generate a score based on their intensity. This score can predict the stages of cancer progression in patients through glycan characterization. This methodology offers a potential means of diagnosing lung cancer through serum glycome analysis.

102-Plex Approach for Accurate and Multiplexed Proteome Quantification.

Hyperplexing approaches have been aimed to meet the demand for large-scale proteomic analyses. Currently, the analysis capacity has expanded to up to 54 samples within a single experiment by utilizing different isotopic and isobaric reagent combinations. In this report, we propose a super multiplexed approach to enable the analysis of up to 102 samples in a single experiment, by the combination of our recently developed TAG-TMTpro and TAG-IBT16 labeling. We systematically investigated the identification and quantification performance of the 102-plex approach using the mixtures of E. coli and HeLa peptides. Our results revealed that all labeling series demonstrated accurate and reliable quantification performance. The combination of TAG-IBT16 and TAG-TMTpro approaches expands the multiplexing capacity to 102 plexes, providing a more multiplexed quantification method for even larger-scale proteomic analysis. Data are available via ProteomeXchange with the identifier PXD042398.

Hydrophilic Peptide and Glycopeptide as Immobilized Sorbents for Glycosylation Analysis.

Hydrophilic interaction liquid chromatography (HILIC) is widely used for glycopeptide enrichment in shot-gun glycoproteomics to enhance the glycopeptide signal and minimize the ionization competition of peptides. In this work, we have developed a novel hydrophilic material (glycoHILIC) based on glycopeptides and peptides to provide hydrophilic properties. GlycoHILIC was synthesized by oxidizing cotton and then reacting the resulting aldehyde with the N-terminus of the glycopeptide or peptide by reductive amination. Due to the large amount of hydrophilic carbohydrates and hydrophilic amino acids contained in glycopeptides, glycoHILIC showed significantly better enrichment of glycopeptides than cotton itself. Our results demonstrate that glycoHILIC has high selectivity, a low detection limit, and good stability. Over 257 unique N-linked glycosylation sites in 1477 intact N-glycopeptides from 146 glycoproteins were identified from 1 µL of human serum using glycoHILIC. Serum analysis of pancreatic cancer patients found that 38 N-glycopeptides among 21 glycoproteins changed significantly, of which 7 N-glycopeptides increased and 31 N-glycopeptides decreased. These results demonstrate that glycoHILIC can be used for glycopeptide enrichment and analysis.

Hyperplexing Approaches for up to 45-Plex Quantitative Proteomic Analysis.

Isobaric labeling has emerged as an indispensable quantitative proteomic approach for its unprecedented multiplexing capacity in a single analysis. Currently, different hyperplexing approaches have been developed to meet the demand for the increasing sample size in large-scale cohort analysis. In this report, we present a tribrid hyperplexing approach by the combinatorial use of three types of isobaric reagents, a novel isobaric tag 16-plex (IBT16) reagent and the widely used tandem mass tag (TMT; TMT11) and TMTpro (TMT18) reagents. After the determination of labeling efficiency and the optimization of testing conditions, we systematically evaluated the identification and quantification performance of the three labeling reagents in both independent and combinatorial manners using the mixtures of E. coli and HeLa peptides with different ratios. Our results reveal that the three reagents are quite similar in all testing aspects despite some differences, and the combination use of the three reagents could expand the multiplexing capacity to up to 45-plex. Furthermore, we conclude the advantages of IBT16 in the combination use and the preferred combinations for different practical applications. Data are available via ProteomeXchange with identifier PXD037498.

Novel Approach to Enriching Glycosylated RNAs: Specific Capture of GlycoRNAs via Solid-Phase Chemistry.

Ribonuclease (RNA) modifications can alter cellular function and lead to differential immune responses by acting as discriminators between RNAs from different phyla. RNA glycosylation has recently been observed at the cell surface, and its dysregulation in disease may change RNA functions. However, determining which RNA substrates can be glycosylated remains to be explored. Here, we develop a solid-phase chemoenzymatic method (SPCgRNA) for targeting glycosylated RNAs, by which glycosylated RNA substrates can be specifically recognized. We found the differential N-glycosylation of small RNAs in hTERT-HPNE and MIA PaCa-2 cancer cells using SPCgRNA. RNA-Seq showed that the changes in glyco-miRNAs prepared from SPCgRNA were consistent with those of traditional methods. The KEGG signaling pathway analysis revealed that differential miRNA glycosylation can affect tumor cell proliferation and survival. Further studies found that NGI-1 significantly inhibited the proliferation, migration, and circulation of MIA PaCa-2 and promoted cell apoptosis. In addition, ß-1,4-galactosyltransferase 1 (B4GALT1) not only affected the expression level of glycosylated miRNAs hsa-miR-21-5p but also promoted cell apoptosis and inhibited the cell cycle possibly through the p53 signaling pathway, while B4GALT1 and p53 were also affected following the hsa-miR-21-5p increase. These results suggest that B4GALT1 may catalyze miRNAs glycosylation, which further promotes cancer cell progression.

An analysis of antithrombotic therapy in elderly patients with atrial fibrillation undergoing percutaneous coronary interventions.

As a result of large, randomized trials and updates to clinical guidelines, antithrombotic therapy following percutaneous coronary intervention (PCI) has changed in recent years for patients with nonvalvular atrial fibrillation (NVAF). The purpose of this study was to investigate the real-world data of antithrombotic regimens at discharge and their evolving trends, as well as compare the effect of different therapies on the incidence of major cardiovascular and cerebrovascular ischemic events (MACCEs) and bleeding events in elderly patients. An analysis of 6298 stent implantation patients from 2016 to 2018 was carried out retrospectively. Atrial fibrillation (AF) patients ages 65 and older were divided into two groups according to the antithrombotic regimens prescribed at hospital discharge: dual antiplatelet aggregation treatment group (DAPT) and anticoagulant treatment and antiplatelet aggregation treatment group (ATT). Baseline characteristics, efficacy endpoints (MACCEs/cerebrovascular ischemic events) and safety endpoints (bleeding events) were analysed and compared between the different antithrombotic regiments. During 2016 to 2018, the use of oral anticoagulants (OAC) increased from 16.3% to 54.1% (p trend <0.01). Since the introduction of non-vitamin K antagonist oral anticoagulants (NOACs), warfarin usage has decreased from 100% to 41.7%, and NOACs have rapidly replaced warfarin. The rate of persistent AF in the ATT group was significantly higher than the rate in the DAPT group (79.6% vs 59.7%, p = 0.01), and the ATT group used more proton pump inhibitors (PPI) than the DAPT group (23.3% vs 11.8%, p = 0.01). A significant decrease was observed in MACCEs (10.7% vs 26.0%, p < 0.01) and cerebrovascular ischemic events (2.9% vs 11.8%, p = 0.01) in the ATT group compared with the DAPT group. According to the ATT subgroup analysis, there was a significant difference in the incidence of overall bleeding between the triple anticoagulant therapy group and the dual anticoagulant therapy group (DT) (18.0% vs 2.4%, p = 0.02). MACCEs were predicted independently by ATT and CHA2 DS2 -VASc (congestive heart failure, hypertension, age ≥75 years, diabetes, stroke or transient ischemic attack, vascular disease, age 65 to 74 years, sex category) scores, whereas bleeding was predicted independently by PPI use and HAS-BLED (hypertension, abnormal renal/liver function, stroke, bleeding history or predisposition, labile international normalized ratio, elderly, drugs/alcohol) scores. As a result of NOAC introduction and use, the combination of antithrombotic regimens at discharge for elderly patients with AF after PCI has changed rapidly over the past few years toward a higher use of ATTs, whereas patients with AF undergoing PCI still rarely receive an appropriate antithrombotic regimen. It is essential to conduct ATT in elderly patients who are undergoing PCI, and further DT may be more appropriate.

TAG-TMTpro, a Hyperplexing Quantitative Approach for High-Throughput Proteomic Studies.

Isobaric labeling is the most widely used multiplexing quantitative approach in proteomic studies, enabling the comparison of up to 18 samples in a single MS analysis. Expanding the multiplexing capacity is of great necessity for high-throughput proteomic studies. Herein, we establish a novel TAG-TMTpro approach by introducing Ala or Gly residues to peptides prior to TMTpro labeling, which is able to triple the quantitative capacity of TMTpro. We systematically evaluated the Boc-Ala-OSu and Boc-Gly-OSu reaction and optimized the conditions for labeling, side-product elimination, and Boc deprotection. We validated the identification and quantification performance using E. coli and HeLa cell lysates. We demonstrated that the TAG-TMTpro approach resulted in good identification reproducibility and reliable quantitative accuracy. The TAG-TMTpro is able to triple the multiplexing capacity of TMTpro reagents and is a versatile quantitative approach for high-throughput proteomic studies. Data are available via ProteomeXchange with identifier PXD033711.

Phosphorylation of Msx1 promotes cell proliferation through the Fgf9/18-MAPK signaling pathway during embryonic limb development.

Msh homeobox (Msx) is a subclass of homeobox transcriptional regulators that control cell lineage development, including the early stage of vertebrate limb development, although the underlying mechanisms are not clear. Here, we demonstrate that Msx1 promotes the proliferation of myoblasts and mesenchymal stem cells (MSCs) by enhancing mitogen-activated protein kinase (MAPK) signaling. Msx1 directly binds to and upregulates the expression of fibroblast growth factor 9 (Fgf9) and Fgf18. Accordingly, knockdown or antibody neutralization of Fgf9/18 inhibits Msx1-activated extracellular signal-regulated kinase 1/2 (Erk1/2) phosphorylation. Mechanistically, we determined that the phosphorylation of Msx1 at Ser136 is critical for enhancing Fgf9 and Fgf18 expression and cell proliferation, and cyclin-dependent kinase 1 (CDK1) is apparently responsible for Ser136 phosphorylation. Furthermore, mesenchymal deletion of Msx1/2 results in decreased Fgf9 and Fgf18 expression and Erk1/2 phosphorylation, which leads to serious defects in limb development in mice. Collectively, our findings established an important function of the Msx1-Fgf-MAPK signaling axis in promoting cell proliferation, thus providing a new mechanistic insight into limb development.

Multi-Omics Analysis in ß-Thalassemia Using an HBB Gene-Knockout Human Erythroid Progenitor Cell Model.

ß-thalassemia is a hematologic disease that may be associated with significant morbidity and mortality. Increased expression of HBG1/2 can ameliorate the severity of ß-thalassemia. Compared to the unaffected population, some ß-thalassemia patients display elevated HBG1/2 expression levels in their red blood cells. However, the magnitude of up-regulation does not reach the threshold of self-healing, and thus, the molecular mechanism underlying HBG1/2 expression in the context of HBB-deficiency requires further elucidation. Here, we performed a multi-omics study examining chromatin accessibility, transcriptome, proteome, and phosphorylation patterns in the HBB homozygous knockout of the HUDEP2 cell line (HBB-KO). We found that up-regulation of HBG1/2 in HBB-KO cells was not induced by the H3K4me3-mediated genetic compensation response. Deletion of HBB in human erythroid progenitor cells resulted in increased ROS levels and production of oxidative stress, which led to an increased rate of apoptosis. Furthermore, in response to oxidative stress, slower cell cycle progression and proliferation were observed. In addition, stress erythropoiesis was initiated leading to increased intracellular HBG1/2 expression. This molecular model was also validated in the single-cell transcriptome of hematopoietic stem cells from ß-hemoglobinopathy patients. These findings further the understanding of HBG1/2 gene regulatory networks and provide novel clinical insights into ß-thalassemia phenotypic diversity.

A large-scale group decision making method to select the ideal mobile health application for the hospital.

Mobile health, which is not limited by time and space, can effectively alleviate the imbalance of medical resources. Currently, more and more hospitals begin to pay attention to online medical care and actively expand their mobile channels. Among of which, the cooperation with the third-party platform is an effective way to expand the online services of most hospitals. With the increasing number of mobile health applications (mHealth apps), it is difficult to select the ideal application. Most of the existing studies on mHealth app selection are conducted from the perspective of users who have health needs, which is insufficient. The views of multiple stakeholders should be taken into account. mHealth app selection can be regarded as a large-scale group decision making (LSGDM) problem. In this paper, a hybrid LSGDM method is proposed to select the mHealth app with the highest user satisfaction. First, the weights of criteria are obtained based on quality function deployment and 2-additive measure. Furthermore, a consensus model that considers cooperative and non-cooperative behaviors of decision makers is applied to select the ideal mHealth app. Finally, an illustrative example is implemented to exhibit the utility and validity of the proposed model.

[Preparation and in vitro evaluation of quercetin nanosuspension stabilized by gypenosides].

This study was designed to explore the potential of gypenosides as a novel natural stabilizer for the production of nanosuspensions. The gypenosides-stabilized quercetin nanosuspensions(QUE-NS) were prepared using the high-speed shearing and high-pressure homogenization method with quercetin as a model drug, followed by their in vitro evaluation.Based on the measured mean particle size and polydispersity index(PDI) of QUE-NS,the single factor experiment was conducted to optimize the preparation process parameters.The freeze-drying method was used to transform QUE-NS into freeze-dried powders, whose storage stability and saturation solubility were then studied.Moreover, the effects of pH and ionic strength on the physical stability of the nanosuspension system were examined.According to the results, the optimized process parameters were listed as follows: shear rate 13 000 r·min~(-1),shear time 2 min, homogenization pressure 100 MPa, and homogenization frequency 12 times.The mean particle size of QUE-NS prepared under the optimum process conditions was(461.9±2.4) nm, and the PDI was 0.059±0.016.During the two months of storage at room temperature, the freeze-dried QUE-NS powders remained stable.The saturation solubility of freeze-dried QUE-NS powders was proved higher than those of quercetin and the physical mixture.The results of stability testing demonstrated that QUE-NS stabilized with gypenosides exhibited good stability within the pH range of 6 to 8,while coalescence was prone to occur in the presence of salt.Overall, gypenosides is expected to become a new natural stabilizer for the preparation of nanosuspensions.

Tyrosine-EDC Conjugation, an Undesirable Side Effect of the EDC-Catalyzed Carboxyl Labeling Approach.

Carbodiimide-catalyzed carboxyl and amine conjugation (amidation) has been widely used to protect carboxyl groups. N-(3-(Dimethylamino)propyl)-N'-ethylcarbodiimide (EDC) is the most common carbodiimide reagent in protein chemistry due to its high catalytic efficiency in aqueous media. The reaction has also been applied in different proteomic studies including protein terminomics, glycosylation, and interaction. Herein, we report that the EDC-catalyzed amidation could cause a +155 Da side modification on the tyrosine residue and severely hamper the identification of Tyr-containing peptides. We revealed the extremely low identification rate of Tyr-containing peptides in different published studies employing the EDC-catalyzed amidation. We discovered a +155 Da side modification occurring specifically on Tyr and decoded it as the addition of EDC. Consideration of the side modification in a database search enabled the identification of 13 times more Tyr-containing peptides. Furthermore, we successfully developed an efficient method to remove the side modification. Our results also imply that chemical reactions in proteomic studies should be carefully evaluated prior to their wide applications. Data are available via ProteomeXchange with identifier PXD020042.

Msx1 cooperates with Runx1 for inhibiting myoblast differentiation.

Myogenesis is an important and complicated biological process, especially during the process of embryonic development. The homeoprotein Msx1 is a crucial transcriptional repressor of myogenesis and maintains myogenic precursor cells in an undifferentiated, proliferative state. However, the molecular mechanism through which Msx1 coordinates myogenesis remains to be elucidated. Here, we determine the interacting partner proteins of Msx1 in myoblast cells by a proteomic screening method. Msx1 is found to interact with 55 proteins, among which our data demonstrate that the cooperation of Runt-related transcription factor 1 (Runx1) with Msx1 is required for myoblast cell differentiation. Our findings provide important insights into the mechanistic roles of Msx1 in myoblast cell differentiation, and lays foundation for the myogenic differentiation process.

Lupeol and its derivatives as anticancer and anti-inflammatory agents: Molecular mechanisms and therapeutic efficacy.

Lupeol is a natural triterpenoid that widely exists in edible fruits and vegetables, and medicinal plants. In the last decade, a plethora of studies on the pharmacological activities of lupeol have been conducted and have demonstrated that lupeol possesses an extensive range of pharmacological activities such as anticancer, antioxidant, anti-inflammatory, and antimicrobial activities. Pharmacokinetic studies have indicated that absorption of lupeol by animals was rapid despite its nonpolar characteristics, and lupeol belongs to class II BCS (biopharmaceutics classification system) compounds. Moreover, the bioactivities of some isolated or synthesized lupeol derivatives have been investigated, and these results showed that, with modification to C-3 or C-19, some derivatives exhibit stronger activities, e.g., antiprotozoal or anticancer activity. This review aims to summarize the advances in pharmacological and pharmacokinetic studies of lupeol in the last decade with an emphasis on its anticancer and anti-inflammatory activities, as well as the research progress of lupeol derivatives thus far, to provide researchers with the latest information, point out the limitations of relevant research at the current stage and the aspects that should be strengthened in future research.

The antihypertensive potential of flavonoids from Chinese Herbal Medicine: A review.

With the coming of the era of the aging population, hypertension has become a global health burden to be dealt with. Although there are multiple drugs and procedures to control the symptoms of hypertension, the management of it is still a long-term process, and the side effects of conventional drugs pose a burden on patients. Flavonoids, common compounds found in fruits and vegetables as secondary metabolites, are active components in Chinese Herbal Medicine. The flavonoids are proved to have cardiovascular benefits based on a plethora of animal experiments over the last decade. Thus, the flavonoids or flavonoid-rich plant extracts endowed with anti-hypertension activities and probable mechanisms were reviewed. It has been found that flavonoids may affect blood pressure in various ways. Moreover, despite the substantial evidence of the potential for flavonoids in the control of hypertension, it is not sufficient to support the clinical application of flavonoids as an adjuvant or core drug. So the synergistic effects of flavonoids with other drugs, pharmacokinetic studies, clinical trials and the safety of flavonoids are also incorporated in the discussion. It is believed that more breakthrough studies are needed. Overall, this review may shed some new light on the explicit recognition of the mechanisms of anti-hypertension actions of flavonoids, pointing out the limitations of relevant research at the current stage and the aspects that should be strengthened in future researches.

Bergapten: A review of its pharmacology, pharmacokinetics, and toxicity.

Bergapten is a natural furocoumarin, also known as 5-methoxypsoralen, and its medicinal value has been paid more and more attention. By sorting out the pharmacological literature of bergapten, we found that bergapten has a wide range of pharmacological effects, including neuroprotection, organ protection, anticancer, antiinflammatory, antimicrobial, and antidiabetes effects. However，bergapten has complex impacts on the hepatic metabolic enzyme. Moreover, pharmacokinetic studies showed that bergapten has higher absolute bioavailability and can cross the blood-brain barrier and has a great potential for treating brain disease, but the mechanism needs further clarification to make greater use of its ability to treat brain diseases. Furthermore, the phototoxicity of bergapten combined with ultraviolet light has always been mentioned. In view of its wide range of pharmacological activities, bergapten is expected to be a potential drug candidate for the treatment of diabetes and diabetes-induced osteoporosis, epilepsy, Alzheimer's disease, depression, and cancer. However, further studies are needed to elucidate its molecular mechanisms and targets. The phototoxicity of bergapten as a side effect should be further avoided. On the other hand, the photoactivation of bergapten in the anticancer aspect can be better utilized.

Formaldehyde Derivatization: An Unexpected Side Reaction During Filter-Aided Sample Preparation.

The filter-aided sample preparation (FASP) method has been commonly used for proteomic sample preparation due to its high efficiency in removing impurities. Herein, we report an overlooked +12 Da side modification during FASP method using Microcon spin filters. We confirmed that the side modification is caused by formaldehyde released from the spin filter and found that the side modification leads to 10.5% and 9.5% loss in proteome-level peptide and protein identification, respectively. We evaluated different pretreatment procedures to reduce the side reaction. Furthermore, on the basis of the evaluation results of different brands of spin filters, we recommend Nanosep spin filters for different proteomic studies, especially for amine-labeling proteomic studies. Our results would benefit researchers employing the spin filters to improve their results and also help spin filter manufacturers to improve the product quality. Data are available via ProteomeXchange with identifier PXD018737.

Basic Strong Cation Exchange Chromatography, BaSCX, a Highly Efficient Approach for C-Terminomic Studies Using LysargiNase Digestion.

Decoding protein C-termini is a challenging task in protein chemistry using conventional chemical and enzymatic approaches. With the rapid development in modern mass spectrometer, many advanced mass spectrometry (MS)-based protein C-termini analysis approaches have been established. Although great progress is being continually achieved, it is still necessary to develop more efficient approaches in order to discover a proteome-scale protein C-termini (C-terminome) and consequently to help understand their biological functions. In this report, we describe the BaSCX method, for basic strong cation exchange chromatography, for C-terminome studies. Taking advantage of carboxylic amidation, LysargiNase digestion, and optimized search parameters, BaSCX enables identification of 1806 and 1812 database-annotated human protein C-termini from HeLa and 293T cells, resepctively, by triplicate experiments using 40 µg proteins each. Combined together, 2151 human protein C-termini, nearly three times the recently reported largest human C-terminome data set, are reported in this study. Similar results were acquired in different organisms, including mice, C. elegans, and tomatoes. Furthermore, we report for the first time the discovery of C-terminal-specific modifications using a proteomic approach, including three methyl-esterified protein C-termini and 16 α-amidated protein C-termini, demonstrating the excellent performance and great potential of BaSCX in C-terminomic studies. Data are available via ProteomeXchange with identifier PXD016317.

Short-Term Mild Temperature-Stress-Induced Alterations in the C. elegans Phosphoproteome.

Exposure to mild early-life stresses can slow down aging, and protein phosphorylation might be an essential regulator in this process. However, the mechanisms of phosphorylation-based signaling networks during mild early-life stress remain elusive. Herein, we systematically analyzed the phosphoproteomes of Caenorhabditis elegans, which were treated with three mild temperatures (15 °C, 20 °C, and 25 °C) in two different short-term groups (10 min and 60 min). By utilizing an iTRAQ-based quantitative phosphoproteomic approach, 18,187 phosphosites from 3330 phosphoproteins were detected in this study. Volcano plots illustrated that the phosphorylation abundance of 374 proteins and 347 proteins, were significantly changed at 15 °C and 25 °C, respectively. Gene ontology, KEGG pathway and protein-protein interaction network analyses revealed that these phosphoproteins were primarily associated with metabolism, translation, development, and lifespan determination. A motif analysis of kinase substrates suggested that MAPK, CK, and CAMK were most likely involved in the adaption processes. Moreover, 16 and 14 aging-regulated proteins were found to undergo phosphorylation modifications under the mild stresses of 15 °C and 25 °C, respectively, indicating that these proteins might be important for maintaining long-term health. Further lifespan experiments confirmed that the candidate phosphoproteins, e.g., EGL-27 and XNP-1 modulated longevity at 15 °C, 20 °C, and 25 °C, and they showed increased tolerance to thermal and oxidative stresses. In conclusion, our findings offered data that supports understanding of the phosphorylation mechanisms involved in mild early-life stresses in C. elegans. Data are available via ProteomeXchange with identifier PXD021081.

Global Quantitative Proteomics Studies Revealed Tissue-Preferential Expression and Phosphorylation of Regulatory Proteins in Arabidopsis.

Organogenesis in plants occurs across all stages of the life cycle. Although previous studies have identified many genes as important for either vegetative or reproductive development at the RNA level, global information on translational and post-translational levels remains limited. In this study, six Arabidopsis stages/organs were analyzed using quantitative proteomics and phosphoproteomics, identifying 2187 non-redundant proteins and evidence for 1194 phosphoproteins. Compared to the expression observed in cauline leaves, the expression of 1445, 1644, and 1377 proteins showed greater than 1.5-fold alterations in stage 1-9 flowers, stage 10-12 flowers, and open flowers, respectively. Among these, 294 phosphoproteins with 472 phosphorylation sites were newly uncovered, including 275 phosphoproteins showing differential expression patterns, providing molecular markers and possible candidates for functional studies. Proteins encoded by genes preferentially expressed in anther (15), meiocyte (4), or pollen (15) were enriched in reproductive organs, and mutants of two anther-preferentially expressed proteins, acos5 and mee48, showed obviously reduced male fertility with abnormally organized pollen exine. In addition, more phosphorylated proteins were identified in reproductive stages (1149) than in the vegetative organs (995). The floral organ-preferential phosphorylation of GRP17, CDC2/CDKA.1, and ATSK11 was confirmed with western blot analysis. Moreover, phosphorylation levels of CDPK6 and MAPK6 and their interacting proteins were elevated in reproductive tissues. Overall, our study yielded extensive data on protein expression and phosphorylation at six stages/organs and provides an important resource for future studies investigating the regulatory mechanisms governing plant development.