RNF152 Suppresses Fatty Acid Oxidation and Metastasis of Lung Adenocarcinoma by Inhibiting IRAK1-Mediated AKR1B10 Expression.

Lung adenocarcinoma (LUAD) is a common subtype of primary lung cancer. Fatty acid oxidation plays a key role in LUAD development by providing energy for tumor cells. This study aimed to identify the role of ring finger protein 152 (RNF152) in LUAD. RNF152 was down-regulated in LUAD, and low RNF152 expression correlated with a poor prognosis in LUAD patients. RNF152 overexpression inhibited the proliferation and malignant phenotype of LUAD cells, whereas RNF152 knockdown exerted an opposite effect. Tumor cells overexpressing RNF152 showed less fatty acid oxidation compared with control cells, whereas RNF152 knockdown induced fatty acid uptake and oxidation. Further analysis revealed the binding reaction between RNF152 and interleukin-1 receptor-associated kinase 1 (IRAK1). RNF152 reduced the stability of IRAK1 in LUAD cells by promoting its ubiquitination. RNF152-overexpressed tumor cells exhibited a significantly lower level of Aldo-Keto reductase family 1 member 10 (AKR1B10), whereas up-regulation of IRAK1 restored the expression of AKR1B10 in RNF152-overexpressed cells. Furthermore, up-regulation of IRAK1 eliminated the antitumor effect of RNF152 in LUAD cells. Mouse xenograft models confirmed the inhibitory effect of RNF152 on the tumorigenesis and metastasis of LUAD. Taken together, RNF152 played a tumor suppressive role in LUAD by promoting IRAK1 ubiquitination and IRAK1-mediated down-regulation of AKR1B10, thereby reversing the malignant phenotype of LUAD.

Recent advances (2019-2021) of capillary electrophoresis-mass spectrometry for multilevel proteomics.

Multilevel proteomics aims to delineate proteins at the peptide (bottom-up proteomics), proteoform (top-down proteomics), and protein complex (native proteomics) levels. Capillary electrophoresis-mass spectrometry (CE-MS) can achieve highly efficient separation and highly sensitive detection of complex mixtures of peptides, proteoforms, and even protein complexes because of its substantial technical progress. CE-MS has become a valuable alternative to the routinely used liquid chromatography-mass spectrometry for multilevel proteomics. This review summarizes the most recent (2019-2021) advances of CE-MS for multilevel proteomics regarding technological progress and biological applications. We also provide brief perspectives on CE-MS for multilevel proteomics at the end, highlighting some future directions and potential challenges.

Changes in soil bacterial community and functions by substituting chemical fertilizer with biogas slurry in an apple orchard.

Growing concerns about the negative environmental effects of excessive chemical fertilizer input in fruit production have resulted in many attempts looking for adequate substitution. Biogas slurry as a representative organic fertilizer has the potential to replace chemical fertilizer for improvement of sustainability. However, it is still poorly known how biogas slurry applications may affect the composition of soil microbiome. Here, we investigated different substitution rates of chemical fertilizer with biogas slurry treatment (the control with no fertilizer and biogas slurry, CK; 100% chemical fertilizer, CF; biogas slurry replacing 50% of chemical fertilizer, CBS; and biogas slurry replacing 100% of chemical fertilizer, BS) in an apple orchard. Soil bacterial community and functional structure among treatments were determined using Illumina sequencing technology coupled with Functional Annotation of Prokaryotic Taxonomy (FAPROTAX) analysis. Leaf nutrient contents, apple fruit and soil parameters were used to assess plant and soil quality. Results showed that most of fruit parameters and soil properties were significantly varied in the four treatments. CBS treatment increased the contents of soil organic matter, alkali nitrogen and available potassium average by 49.8%, 40.7% and 27.9%, respectively. Treatments with biogas slurry application increased the single fruit weight, fresh weight, and dry weight of apple fruit average by 15.6%, 18.8% and 17.8, respectively. Soil bacterial community dominance and composition were significantly influenced by substituting of chemical fertilizer with biogas slurry. Biogas slurry application enhanced the relative abundance of some beneficial taxa (e.g. Acidobacteria Gp5 and Gp7, Parasegetibacter) and functional groups related to carbon and nitrogen cycling such as chemoheterotrophy, cellulolysis, and nitrogen fixation. Soil available phosphorus and potassium, pH and electrical conductivity were identified having a high potential for regulating soil bacterial specific taxa and functional groups. This study showed that the proper ratio application (50%: 50%) of biogas slurry with chemical fertilizer could regulate soil bacterial composition and functional structure via changes in soil nutrients. The variations of bacterial community could potentially take significant ecological roles in maintaining apple plant growth, soil fertility and functionality.

Membrane Ultrafiltration-Based Sample Preparation Method and Sheath-Flow CZE-MS/MS for Top-Down Proteomics.

Mass spectrometry (MS)-based denaturing top-down proteomics (dTDP) identify proteoforms without pretreatment of enzyme proteolysis. A universal sample preparation method that can efficiently extract protein, reduce sample loss, maintain protein solubility, and be compatible with following up liquid-phase separation, MS, and tandem MS (MS/MS) is vital for large-scale proteoform characterization. Membrane ultrafiltration (MU) was employed here for buffer exchange to efficiently remove the sodium dodecyl sulfate (SDS) detergent in protein samples used for protein extraction and solubilization, followed by capillary zone electrophoresis (CZE)-MS/MS analysis. The MU method showed good protein recovery, minimum protein bias, and nice compatibility with CZE-MS/MS. Single-shot CZE-MS/MS analysis of an Escherichia coli sample prepared by the MU method identified over 800 proteoforms.

Protein disulfide isomerases (PDIs) negatively regulate ebolavirus structural glycoprotein expression in the endoplasmic reticulum (ER) via the autophagy-lysosomal pathway.

Zaire ebolavirus (EBOV) causes a severe hemorrhagic fever in humans and non-human primates with high morbidity and mortality. EBOV infection is dependent on its structural glycoprotein (GP), but high levels of GP expression also trigger cell rounding, detachment, and downregulation of many surface molecules that is thought to contribute to its high pathogenicity. Thus, EBOV has evolved an RNA editing mechanism to reduce its GP expression and increase its fitness. We now report that the GP expression is also suppressed at the protein level in cells by protein disulfide isomerases (PDIs). Although PDIs promote oxidative protein folding by catalyzing correct disulfide formation in the endoplasmic reticulum (ER), PDIA3/ERp57 adversely triggered the GP misfolding by targeting GP cysteine residues and activated the unfolded protein response (UPR). Abnormally folded GP was targeted by ER-associated protein degradation (ERAD) machinery and, unexpectedly, was degraded via the macroautophagy/autophagy-lysosomal pathway, but not the proteasomal pathway. PDIA3 also decreased the GP expression from other ebolavirus species but increased the GP expression from Marburg virus (MARV), which is consistent with the observation that MARV-GP does not cause cell rounding and detachment, and MARV does not regulate its GP expression via RNA editing during infection. Furthermore, five other PDIs also had a similar inhibitory activity to EBOV-GP. Thus, PDIs negatively regulate ebolavirus glycoprotein expression, which balances the viral life cycle by maximizing their infection but minimizing their cellular effect. We suggest that ebolaviruses hijack the host protein folding and ERAD machinery to increase their fitness via reticulophagy during infection.Abbreviations: 3-MA: 3-methyladenine; 4-PBA: 4-phenylbutyrate; ACTB: ß-actin; ATF: activating transcription factor; ATG: autophagy-related; BafA1: bafilomycin A1; BDBV: Bundibugyo ebolavirus; CALR: calreticulin; CANX: calnexin; CHX: cycloheximide; CMA: chaperone-mediated autophagy; ConA: concanamycin A; CRISPR: clusters of regularly interspaced short palindromic repeats; Cas9: CRISPR-associated protein 9; dsRNA: double-stranded RNA; EBOV: Zaire ebolavirus; EDEM: ER degradation enhancing alpha-mannosidase like protein; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; Env: envelope glycoprotein; ER: endoplasmic reticulum; ERAD: ER-associated protein degradation; ERN1/IRE1: endoplasmic reticulum to nucleus signaling 1; GP: glycoprotein; HA: hemagglutinin; HDAC6: histone deacetylase 6; HMM: high-molecular-mass; HIV-1: human immunodeficiency virus type 1; HSPA5/BiP: heat shock protein family A (Hsp70) member 5; IAV: influenza A virus; IP: immunoprecipitation; KIF: kifenesine; Lac: lactacystin; LAMP: lysosomal associated membrane protein; MAN1B1/ERManI: mannosidase alpha class 1B member 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MARV: Marburg virus; MLD: mucin-like domain; NHK/SERPINA1: alpha1-antitrypsin variant null (Hong Kong); NTZ: nitazoxanide; PDI: protein disulfide isomerase; RAVV: Ravn virus; RESTV: Reston ebolavirus; SARS-CoV: severe acute respiratory syndrome coronavirus; SBOV: Sudan ebolavirus; sGP: soluble GP; SQSTM1/p62: sequestosome 1; ssGP: small soluble GP; TAFV: Taï Forest ebolavirus; TIZ: tizoxanide; TGN: thapsigargin; TLD: TXN (thioredoxin)-like domain; Ub: ubiquitin; UPR: unfolded protein response; VLP: virus-like particle; VSV: vesicular stomatitis virus; WB: Western blotting; WT: wild-type; XBP1: X-box binding protein 1.

HIV-1 Nef interacts with the cyclin K/CDK13 complex to antagonize SERINC5 for optimal viral infectivity.

HIV-1-negative factor (Nef) protein antagonizes serine incorporator 5 (SERINC5) by redirecting this potent restriction factor to the endosomes and lysosomes for degradation. However, the precise mechanism remains unclear. Using affinity purification/mass spectrometry, we identify cyclin K (CycK) and cyclin-dependent kinase 13 (CDK13) as a Nef-associated kinase complex. CycK/CDK13 phosphorylates the serine at position 360 (S360) in SERINC5, which is required for Nef downregulation of SERINC5 from the cell surface and its counteractivity of the SERINC5 antiviral activity. To understand the role of S360 phosphorylation, we generate chimeric proteins between CD8 and SERINC5 to study their response to Nef. Nef not only downregulates but, importantly, also binds to this chimera in an S360-dependent manner. Thus, S360 phosphorylation increases interactions between Nef and SERINC5 and initiates the destruction of SERINC5 by the endocytic machinery.

Nanoparticle-Aided Nanoreactor for Nanoproteomics.

Large-scale bottom-up proteomics of few even single cells is crucial for a better understanding of the roles played by cell-to-cell heterogeneity in disease and development. Novel proteomic methodologies with extremely high sensitivity are required for few even single-cell proteomics. Sample processing with high recovery and no contaminants is one key step. Here we developed a nanoparticle-aided nanoreactor for nanoproteomics (Nano3) technique for processing low-nanograms of mammalian cell proteins for proteome profiling. The Nano3 technique employed nanoparticles packed in a capillary channel to form a nanoreactor (≤30 nL) for concentrating, cleaning, and digesting proteins originally in a lysis buffer containing sodium dodecyl sulfate (SDS), followed by nanoRPLC-MS/MS analysis. The Nano3 method identified a 40-times higher number of proteins based on MS/MS from 2-ng mouse brain protein samples compared to the SP3 (single-pot solid-phase-enhanced sample preparation) method, which performed the sample processing using the nanoparticles in a 10 µL solution in an Eppendorf tube. The data indicates a drastically higher sample recovery of the Nano3 compared to the SP3 method for processing mass-limited proteome samples. In this pilot study, the Nano3 method was further applied in processing 10-1000 HeLa cells for bottom-up proteomics, producing 441 ± 263 (n = 4) (MS/MS) and 983 ± 292 (n = 4) [match between runs (MBR)+MS/MS] protein identifications from only 10 HeLa cells using a Q-Exactive HF mass spectrometer. The preliminary results render the Nano3 method a useful approach for processing few mammalian cells for proteome profiling.

Investigating native capillary zone electrophoresis-mass spectrometry on a high-end quadrupole-time-of-flight mass spectrometer for the characterization of monoclonal antibodies.

Native capillary zone electrophoresis-mass spectrometry (CZE-MS) has attracted attentions for the characterization of monoclonal antibodies (mAbs) due to the potential of CZE for highly efficient separations of mAbs under native conditions as well as its compatibility with native electrospray ionization (ESI)-MS. However, the low sample loading capacity and limited separation resolution of native CZE for large proteins and protein complexes (e.g. mAbs) impede the widespread adoption of native CZE-MS. Here, we present a novel native capillary isoelectric focusing (cIEF)-assisted CZE-MS method for the characterization of mAbs with much larger sample loading capacity and significantly better separation resolution than native CZE-MS alone. The native cIEF-assisted CZE-MS employed separation capillaries with a new carbohydrate-based neutral coating, a commercilized electrokinetically pumped sheathflow CE-MS interface, and a high-end quadrupole-time-of-flight (Q-TOF) mass spectrometer. Using the method, we documented the separations of different proteoforms of the SigmaMAb and the detection of its various glyco-proteoforms and homodimer. The native cIEF-assisted CZE-MS separated the NIST mAb into three peaks with a submicroliter sample loading volume, corresponding to its different proteoforms. We observed that both the NIST mAb and its homodimer had eight glyco-proteoforms, four of which had low abundance. The results demonstrate the potential of our native cIEF-assisted CZE-MS method for advancing the characterization of large proteins and protein complexes under native conditions.

Capillary Zone Electrophoresis-Tandem Mass Spectrometry As an Alternative to Liquid Chromatography-Tandem Mass Spectrometry for Top-down Proteomics of Histones.

Top-down proteomics (TDP) is an ideal approach for deciphering the histone code and it routinely employs reversed-phase liquid chromatography (RPLC)-tandem mass spectrometry (MS/MS). Because of the extreme complexity of histones regarding the number of proteoforms, new analytical tools with high-capacity separation and highly sensitive detection of proteoforms are required for TDP of histones. Here we present capillary zone electrophoresis (CZE)-MS/MS via the electro-kinetically pumped sheath-flow CE-MS interface for large-scale top-down delineation of histone proteoforms. CZE-MS/MS identified a comparable number of proteoforms to RPLC-MS/MS from a calf histone sample with more than 30-fold less sample consumption (75-ng vs. Three µg), indicating its substantially higher sensitivity. We identified about 400 histone proteoforms from the calf histone sample using two-dimensional size-exclusion chromatography (SEC)-CZE-MS/MS with less than 300-ng proteins consumed. We identified histone proteoforms carrying various tentative post-translational modifications (PTMs), for example, acetylation, methylation (mono-, di-, and tri-), phosphorylation, and succinylation. The electrophoretic mobility (µef) of unmodified histone proteoforms can be predicted accurately (R2 = 0.98) with an optimized semiempirical model based on our recent work. The results render CZE-MS/MS as a useful tool for deciphering the histone code in a proteoform-specific manner and on a global scale.

Recent technical progress in sample preparation and liquid-phase separation-mass spectrometry for proteomic analysis of mass-limited samples.

Mass spectrometry (MS)-based proteomics has enabled the identification and quantification of thousands of proteins from complex proteomes in a single experiment. However, its performance for mass-limited proteome samples (e.g., single cells and tissue samples from laser capture microdissection) is still not satisfying. The development of novel proteomic methodologies with better overall sensitivity is vital. During the last several years, substantial technical progress has been achieved for the preparation and liquid-phase separation-MS characterization of mass-limited proteome samples. In this review, we summarize recent technological progress of sample preparation, liquid chromatography (LC)-MS, capillary zone electrophoresis (CZE)-MS and MS instrumentation for bottom-up proteomics of trace biological samples, highlight some exciting applications of the novel techniques for single-cell proteomics, and provide a very brief perspective about the field at the end.

Automated Capillary Isoelectric Focusing-Tandem Mass Spectrometry for Qualitative and Quantitative Top-Down Proteomics.

Top-down proteomics (TDP) aims to delineate proteomes in a proteoform-specific manner, which is vital for accurately understanding protein function in cellular processes. It requires high-capacity separation of proteoforms before mass spectrometry (MS) and tandem MS (MS/MS). Capillary isoelectric focusing (cIEF)-MS has been recognized as a useful tool for TDP in the 1990s because cIEF is capable of high-resolution separation of proteoforms. Previous cIEF-MS studies concentrated on measuring the protein's mass without MS/MS, impeding the confident proteoform identification in complex samples and the accurate localization of post-translational modifications on proteoforms. Herein, for the first time, we present automated cIEF-MS/MS-based TDP for large-scale delineation of proteoforms in complex proteomes. Single-shot cIEF-MS/MS identified 711 proteoforms from an Escherichia coli (E. coli) proteome consuming only nanograms of proteins. Coupling two-dimensional size-exclusion chromatography (SEC)-cIEF to ESI-MS/MS enabled the identification of nearly 2000 proteoforms from the E. coli proteome. Label-free quantitative TDP of zebrafish male and female brains using SEC-cIEF-MS/MS quantified thousands of proteoforms and revealed sex-dependent proteoform profiles in brains. Particularly, we discovered several proteolytic proteoforms of pro-opiomelanocortin and prodynorphin with significantly higher abundance in male zebrafish brains as potential endogenous hormone proteoforms. Multilevel quantitative proteomics (TDP and bottom-up proteomics) of the brains revealed that the majority of proteoforms having statistically significant difference in abundance between genders showed no abundance difference at the protein group level. This work represents the first multilevel quantitative proteomics study of sexual dimorphism of the brain.

A bioenergetic shift is required for spermatogonial differentiation.

A bioenergetic balance between glycolysis and mitochondrial respiration is particularly important for stem cell fate specification. It however remains to be determined whether undifferentiated spermatogonia switch their preference for bioenergy production during differentiation. In this study, we found that ATP generation in spermatogonia was gradually increased upon retinoic acid (RA)-induced differentiation. To accommodate this elevated energy demand, RA signaling concomitantly switched ATP production in spermatogonia from glycolysis to mitochondrial respiration, accompanied by increased levels of reactive oxygen species. Disrupting mitochondrial respiration significantly blocked spermatogonial differentiation. Inhibition of glucose conversion to glucose-6-phosphate or pentose phosphate pathway also repressed the formation of c-Kit+ differentiating germ cells, suggesting that metabolites produced from glycolysis are required for spermatogonial differentiation. We further demonstrated that the expression levels of several metabolic regulators and enzymes were significantly altered upon RA-induced differentiation, with both RNA-seq and quantitative proteomic analyses. Taken together, our data unveil a critically regulated bioenergetic balance between glycolysis and mitochondrial respiration that is required for spermatogonial proliferation and differentiation.

Toward a Universal Sample Preparation Method for Denaturing Top-Down Proteomics of Complex Proteomes.

A universal and standardized sample preparation method becomes vital for denaturing top-down proteomics (dTDP) to advance the scale and accuracy of proteoform delineation in complex biological systems. It needs to have high protein recovery, minimum bias, good reproducibility, and compatibility with downstream mass spectrometry (MS) analysis. Here, we employed a lysis buffer containing sodium dodecyl sulfate for extracting proteoforms from cells and, for the first time, compared membrane ultrafiltration (MU), chloroform-methanol precipitation (CMP), and single-spot solid-phase sample preparation using magnetic beads (SP3) for proteoform cleanup for dTDP. The MU method outperformed CMP and SP3 methods, resulting in high and reproducible protein recovery from both Escherichia coli cell (59 ± 3%) and human HepG2 cell (86 ± 5%) samples without a significant bias. Single-shot capillary zone electrophoresis (CZE)-MS/MS analyses of the prepared E. coli and HepG2 cell samples using the MU method identified 821 and 516 proteoforms, respectively. Nearly 30 and 50% of the identified E. coli and HepG2 proteins are membrane proteins. CZE-MS/MS identified 94 histone proteoforms from the HepG2 sample with various post-translational modifications, including acetylation, methylation, and phosphorylation. Our results suggest that combining the SDS-based protein extraction and the MU-based protein cleanup could be a universal sample preparation method for dTDP. The MS raw data have been deposited to the ProteomeXchange Consortium with the data set identifier PXD018248.

Predicting Electrophoretic Mobility of Proteoforms for Large-Scale Top-Down Proteomics.

Large-scale top-down proteomics characterizes proteoforms in cells globally with high confidence and high throughput using reversed-phase liquid chromatography (RPLC)-tandem mass spectrometry (MS/MS) or capillary zone electrophoresis (CZE)-MS/MS. The false discovery rate (FDR) from the target-decoy database search is typically deployed to filter identified proteoforms to ensure high-confidence identifications (IDs). It has been demonstrated that the FDRs in top-down proteomics can be drastically underestimated. An alternative approach to the FDR can be useful for further evaluating the confidence of proteoform IDs after the database search. We argue that predicting retention/migration time of proteoforms from the RPLC/CZE separation accurately and comparing their predicted and experimental separation time could be a useful and practical approach. Based on our knowledge, there is still no report in the literature about predicting separation time of proteoforms using large top-down proteomics data sets. In this pilot study, for the first time, we evaluated various semiempirical models for predicting proteoforms' electrophoretic mobility (µef) using large-scale top-down proteomics data sets from CZE-MS/MS. We achieved a linear correlation between experimental and predicted µef of E. coli proteoforms (R2 = 0.98) with a simple semiempirical model, which utilizes the number of charges and molecular mass of each proteoform as the parameters. Our modeling data suggest that the complete unfolding of proteoforms during CZE separation benefits the prediction of their µef. Our results also indicate that N-terminal acetylation and phosphorylation both decrease the proteoforms' charge by roughly one charge unit.

Comprehensive Detection of Single Amino Acid Variants and Evaluation of Their Deleterious Potential in a PANC-1 Cell Line.

Identifying single amino acid variants (SAAVs) in cancer is critical for precision oncology. Several advanced algorithms are now available to identify SAAVs, but attempts to combine different algorithms and optimize them on large data sets to achieve a more comprehensive coverage of SAAVs have not been implemented. Herein, we report an expanded detection of SAAVs in the PANC-1 cell line using three different strategies, which results in the identification of 540 SAAVs in the mass spectrometry data. Among the set of 540 SAAVs, 79 are evaluated as deleterious SAAVs based on analysis using the novel AssVar software in which one of the driver mutations found in each protein of KRAS, TP53, and SLC37A4 is further validated using independent selected reaction monitoring (SRM) analysis. Our study represents the most comprehensive discovery of SAAVs to date and the first large-scale detection of deleterious SAAVs in the PANC-1 cell line. This work may serve as the basis for future research in pancreatic cancer and personal immunotherapy and treatment.

Improved Nanoflow RPLC-CZE-MS/MS System with High Peak Capacity and Sensitivity for Nanogram Bottom-up Proteomics.

Novel mass spectrometry (MS)-based proteomic tools with extremely high sensitivity and high peak capacity are required for comprehensive characterization of protein molecules in mass-limited samples. We reported a nanoRPLC-CZE-MS/MS system for deep bottom-up proteomics of low micrograms of human cell samples in previous work. In this work, we improved the sensitivity of the nanoRPLC-CZE-MS/MS system drastically via employing bovine serum albumin (BSA)-treated sample vials, improving the nanoRPLC fraction collection procedure, and using a short capillary for fast CZE separation. The improved nanoRPLC-CZE produced a peak capacity of 8500 for peptide separation. The improved system identified 6500 proteins from a MCF7 proteome digest starting with only 500 ng of peptides using a Q-Exactive HF mass spectrometer. The system produced a comparable number of protein identifications (IDs) to our previous system and the two-dimensional (2D) nanoRPLC-MS/MS system developed by Mann's group with 10-fold and 4-fold less sample consumption, respectively. We coupled the single-spot solid phase sample preparation (SP3) method to the improved nanoRPLC-CZE-MS/MS for bottom-up proteomics of 5000 HEK293T cells, resulting in 3689 protein IDs with the consumption of a peptide amount that corresponded to only roughly 1000 cells.

Capillary zone electrophoresis-mass spectrometry for top-down proteomics.

Mass spectrometry (MS)-based top-down proteomics characterizes complex proteomes at the intact proteoform level and provides an accurate picture of protein isoforms and protein post-translational modifications in the cell. The progress of top-down proteomics requires novel analytical tools with high peak capacity for proteoform separation and high sensitivity for proteoform detection. The requirements have made capillary zone electrophoresis (CZE)-MS an attractive approach for advancing large-scale top-down proteomics. CZE has achieved a peak capacity of 300 for separation of complex proteoform mixtures. CZE-MS has shown drastically better sensitivity than commonly used reversed-phase liquid chromatography (RPLC)-MS for proteoform detection. The advanced CZE-MS identified 6,000 proteoforms of nearly 1,000 proteoform families from a complex proteome sample, which represents one of the largest top-down proteomic datasets so far. In this review, we focus on the recent progress in CZE-MS-based top-down proteomics and provide our perspectives about its future directions.

Capillary zone electrophoresis-tandem mass spectrometry for top-down proteomics using attapulgite nanoparticles functionalized separation capillaries.

Attapulgite nanoparticles have good chemical properties and can be modified easily for broad applications. In this work, for the first time, attapulgite nanoparticles were employed to modify the inner wall of separation capillaries for capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS)-based top-down proteomics. The attapulgite nanoparticles and the inner wall of a fused silica capillary were first functionalized with γ-methacryloxypropyl trimethoxysilane. Then the modified nanoparticles and acrylamide were copolymerized in the fused silica capillary with the assistance of azobisisobutyronitrile and heat. The incorporation of high-surface-area nanoparticles in the linear polyacrylamide (LPA) coating resulted in significantly lower electroosmotic mobility compared with the typical LPA coating (3.48 × 10-5 vs. 9.03 × 10-5 cm2 V-1 S-1), most likely because more LPA molecules were immobilized on the inner wall of the separation capillary. The attapulgite nanoparticles functionalized separation capillaries have shown great stability and reproducibility across 43 discontinuous CZE-MS runs of a standard protein mixture. We applied the CZE-MS/MS system for top-down proteomics of Escherichia coli cells. In a proof-of-principle experiment, the CZE-MS/MS system achieved a 90-min separation window and a 1-µL sample loading volume, leading to nearly 300 proteoform and 135 protein identifications in a single run. Many post-translational modifications (PTMs) were identified, including methylation, acetylation, phosphorylation, biotinylation, succinylation, and disulfide bond.

Capillary Zone Electrophoresis-Tandem Mass Spectrometry for Large-Scale Phosphoproteomics with the Production of over 11,000 Phosphopeptides from the Colon Carcinoma HCT116 Cell Line.

Phosphoproteomics requires better separation of phosphopeptides to boost the coverage of the phosphoproteome. We argue that an alternative separation method that produces orthogonal phosphopeptide separation to the widely used LC needs to be considered. Capillary zone electrophoresis (CZE) is one important alternative because CZE and LC are orthogonal for phosphopeptide separation and because the migration time of peptides in CZE can be accurately predicted. In this work, we coupled strong cation exchange (SCX)-reversed-phase LC (RPLC) to CZE-MS/MS for large-scale phosphoproteomics of the colon carcinoma HCT116 cell line. The CZE-MS/MS-based platform identified 11,555 phosphopeptides. The phosphopeptide data set is at least 100% larger than that from previous CZE-MS/MS studies and will be a valuable resource for building a model for predicting the migration time of phosphopeptides in CZE. Phosphopeptides migrate significantly slower than corresponding unphosphopeptides under acidic conditions of CZE separations and in a normal polarity. According to our modeling data, phosphorylation decreases peptide's charge roughly by one charge unit, resulting in dramatic decrease in electrophoretic mobility. Preliminary investigations demonstrate that electrophoretic mobility of phosphopeptides containing one phosphoryl group can be predicted with the same accuracy as for nonmodified peptides ( R2 ≈ 0.99). The CZE-MS/MS and LC-MS/MS were complementary in large-scale phosphopeptide identifications and produced different phosphosite motifs from the HCT116 cell line. The data highlight the value of CZE-MS/MS for phosphoproteomics as a complementary separation approach for not only improving the phosphoproteome coverage but also providing more insight into the phosphosite motifs.

CAD1 and CCR2 protein complex formation in monolignol biosynthesis in Populus trichocarpa.

Lignin is the major phenolic polymer in plant secondary cell walls and is polymerized from monomeric subunits, the monolignols. Eleven enzyme families are implicated in monolignol biosynthesis. Here, we studied the functions of members of the cinnamyl alcohol dehydrogenase (CAD) and cinnamoyl-CoA reductase (CCR) families in wood formation in Populus trichocarpa, including the regulatory effects of their transcripts and protein activities on monolignol biosynthesis. Enzyme activity assays from stem-differentiating xylem (SDX) proteins showed that RNAi suppression of PtrCAD1 in P. trichocarpa transgenics caused a reduction in SDX CCR activity. RNAi suppression of PtrCCR2, the only CCR member highly expressed in SDX, caused a reciprocal reduction in SDX protein CAD activities. The enzyme assays of mixed and coexpressed recombinant proteins supported physical interactions between PtrCAD1 and PtrCCR2. Biomolecular fluorescence complementation and pull-down/co-immunoprecipitation experiments supported a hypothesis of PtrCAD1/PtrCCR2 heterodimer formation. These results provide evidence for the formation of PtrCAD1/PtrCCR2 protein complexes in monolignol biosynthesis in planta.