Mass Spectrometry-Based Top-Down Proteomics in Nanomedicine: Proteoform-Specific Measurement of Protein Corona.

Conventional mass spectrometry (MS)-based bottom-up proteomics (BUP) analysis of the protein corona [i.e., an evolving layer of biomolecules, mostly proteins, formed on the surface of nanoparticles (NPs) during their interactions with biomolecular fluids] enabled the nanomedicine community to partly identify the biological identity of NPs. Such an approach, however, fails to pinpoint the specific proteoforms─distinct molecular variants of proteins in the protein corona. The proteoform-level information could potentially advance the prediction of the biological fate and pharmacokinetics of nanomedicines. Recognizing this limitation, this study pioneers a robust and reproducible MS-based top-down proteomics (TDP) technique for characterizing proteoforms in the protein corona. Our TDP approach has successfully identified about 900 proteoforms in the protein corona of polystyrene NPs, ranging from 2 to 70 kDa, revealing proteoforms of 48 protein biomarkers with combinations of post-translational modifications, signal peptide cleavages, and/or truncations─details that BUP could not fully discern. This advancement in MS-based TDP offers a more advanced approach to characterize NP protein coronas, deepening our understanding of NPs' biological identities. We, therefore, propose using both TDP and BUP strategies to obtain more comprehensive information about the protein corona, which, in turn, can further enhance the diagnostic and therapeutic efficacy of nanomedicine technologies.

Deciphering nanoparticle protein coronas by capillary isoelectric focusing-mass spectrometry-based top-down proteomics.

The nanoparticle (NP) protein corona significantly influences the outcome of nanomedicine. We present the first example of top-down proteomics (TDP) measurement of the protein corona using capillary isoelectric focusing-mass spectrometry, identifying seventy proteoforms of 16 cancer-related genes. This technique has the potential to revolutionize our understanding of the protein corona and advance nanomedicine.

Study on Bulk Texture and Mechanical Properties of As-Extruded Wide Mg-Al-Zn Alloy Sheets with Different Al Addition.

The wide Mg alloy sheets produced by hot extrusion usually can easily form an inhomogeneous texture, resulting in anisotropic mechanical properties and poor formability. However, few studies have been carried out on the bulk texture investigation at different areas of as-extruded Mg alloy sheets, especially the Mg alloys with different alloying elements. In this work, the effect of Al on the bulk texture and mechanical properties at different areas for three wide Mg-Al-Zn alloy sheets with different Al contents (Mg-3Al-0.5Zn, Mg-8Al-0.5Zn and Mg-9Al-0.5Zn) are mainly investigated by neutron diffraction. The results showed that a strong and uneven basal texture was formed in the Mg-3Al-0.5Zn sheet. Meanwhile, the intensity of the basal texture was significantly weakened due to the numerous fine precipitates of Mg17Al12 particles, with the Al content increasing, which hinder the grain growth during extrusion, while fine recrystallized grains have a more random orientation. The enhanced tensile properties in Mg-8Al-0.5Zn and Mg-9Al-0.5Zn alloy sheets are ascribed to the cooperation effect of a refined microstructure, precipitates and weakened basal texture. Among the three Mg alloy sheets, the Mg-8Al-0.5Zn alloy sheet has a yield strength of about 270 MPa, an ultimate tensile strength of about 330 MPa and ultimate elongation of about 16% in the extrusion direction, which possesses the most excellent comprehensive mechanical properties.

Genetic U-Net: Automatically Designed Deep Networks for Retinal Vessel Segmentation Using a Genetic Algorithm.

Recently, many methods based on hand-designed convolutional neural networks (CNNs) have achieved promising results in automatic retinal vessel segmentation. However, these CNNs remain constrained in capturing retinal vessels in complex fundus images. To improve their segmentation performance, these CNNs tend to have many parameters, which may lead to overfitting and high computational complexity. Moreover, the manual design of competitive CNNs is time-consuming and requires extensive empirical knowledge. Herein, a novel automated design method, called Genetic U-Net, is proposed to generate a U-shaped CNN that can achieve better retinal vessel segmentation but with fewer architecture-based parameters, thereby addressing the above issues. First, we devised a condensed but flexible search space based on a U-shaped encoder-decoder. Then, we used an improved genetic algorithm to identify better-performing architectures in the search space and investigated the possibility of finding a superior network architecture with fewer parameters. The experimental results show that the architecture obtained using the proposed method offered a superior performance with less than 1% of the number of the original U-Net parameters in particular and with significantly fewer parameters than other state-of-the-art models. Furthermore, through in-depth investigation of the experimental results, several effective operations and patterns of networks to generate superior retinal vessel segmentations were identified. The codes of this work are available at https://github.com/96jhwei/Genetic-U-Net.

Sensitive and fast characterization of site-specific protein glycosylation with capillary electrophoresis coupled to mass spectrometry.

Glycoproteomic analysis requires efficient separation and sensitive detection to enable the comprehensive characterization of glycan heterogeneity. Here, we report the use of capillary zone electrophoresis-electrospray ionization-mass spectrometry (CZE-ESI-MS) with an electrokinetically-pumped nanospray interface for the study of protein glycosylation microheterogeneity. A fast separation was developed that resolved intact glycopeptides generated from standard proteins within ~9min. Differentially terminal-galactosylated and sialylated species with the same glycosylation sites were well resolved. The concentration detection limits for CZE were three times higher than for nanoLC methods; however, a 200-fold smaller injection volume was used in CZE, which reflects the use of an extremely efficient electrospray interface in our CZE-ESI-MS setup. The resulting glycopeptide mass detection limit was two orders of magnitude superior to a nanoLC method. We also observed a 1.5% and 7% average relative standard deviation in peak migration time and glycopeptide relative abundance, and a four order of magnitude linear dynamic range in signal intensity. With CZE-ESI-MS, 40 haptoglobin glycopeptides were identified from roughly 40 fmol of digest.

Embedded vertically aligned cadmium telluride nanorod arrays grown by one-step electrodeposition for enhanced energy conversion efficiency in three-dimensional nanostructured solar cells.

Vertically aligned CdTe nanorods (NRs) arrays are successfully grown by a simple one-step and template-free electrodeposition method, and then embedded in the CdS window layer to form a novel three-dimensional (3D) heterostructure on flexible substrates. The parameters of electrodeposition such as deposition potential and pH of the solution are varied to analyze their important role in the formation of high quality CdTe NRs arrays. The photovoltaic conversion efficiency of the solar cell based on the 3D heterojunction structure is studied in detail. In comparison with the standard planar heterojunction solar cell, the 3D heterojunction solar cell exhibits better photovoltaic performance, which can be attributed to its enhanced optical absorption ability, increased heterojunction area and improved charge carrier transport. The better photoelectric property of the 3D heterojunction solar cell suggests great application potential in thin film solar cells, and the simple electrodeposition process represents a promising technique for large-scale fabrication of other nanostructured solar energy conversion devices.

Synthesis of uniform cadmium sulphide thin film by the homogeneous precipitation method on cadmium telluride nanorods and its application in three-dimensional heterojunction flexible solar cells.

High-density CdTe nanorod arrays are successfully embedded in a uniform and compact CdS layer, forming a novel three-dimensional (3D) CdTe NRs/CdS heterojunction structure. The CdS layer is prepared by homogeneous precipitation (HP) method using decomposition of urea. The effects of temperature and concentration of reactants on the growth and composition of CdS film are investigated in detail. The results demonstrate that the temperature affects the thermal decomposition of urea significantly, and the concentration of CdCl2 and CS (NH2)2 plays an essential role in the compositional ratio of CdS film. Further investigations reveal that, in comparison with the traditional precipitation method, a better coverage of CdS on the surface of CdTe NRs can be obtained by HP method due to the slow and even hydrolysis of urea. Moreover, photovoltaic performance of the novel CdTe NRs/CdS 3D photovoltaic device is also investigated. This study demonstrates that the 3D heterostructure has potential application in thin film solar cells, and the successful deposition of CdS layer on the surface of CdTe NRs by HP method suggests a promising technique for large-scale fabrication of these solar cells.

Simplified capillary isoelectric focusing with chemical mobilization for intact protein analysis.

We report a capillary isoelectric focusing system based on a sequential injection method for simplified chemical mobilization. This system was coupled to an ion trap mass spectrometer with an electrokinetically pumped nanoelectrospray interface. The nanoelectrospray emitter employed an acidic sheath electrolyte. To simplify focusing and mobilization, a plug of ammonium hydroxide was first injected into the capillary, followed by a section of mixed sample and ampholyte. During focusing, the NH3 H2 O section worked as catholyte. As focusing progressed, the NH3 H2 O section was titrated to lower pH by the acidic sheath electrolyte. Chemical mobilization started automatically once the ammonium hydroxide was consumed by the acidic sheath flow electrolyte, which then acted as the mobilization solution. In this report, the lengths of the NH3 H2 O section and sample were optimized. With a 1 m long capillary, a relative short plug of the NH3 H2 O section (3 cm) produced both fast migration and reasonable separation resolution. The simplified capillary isoelectric focusing mass spectrometry system produced base peak intensity relative standard deviation of 8.5% and migration time relative standard deviation ≤0.6% for myoglobin and cytochrome C in triplicate runs.

Over 4100 protein identifications from a Xenopus laevis fertilized egg digest using reversed-phase chromatographic prefractionation followed by capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry analysis.

A tryptic digest generated from Xenopus laevis fertilized embryos was fractionated by RPLC. One set of 30 fractions was analyzed by 100-min CZE-ESI-MS/MS separations (50 h total instrument time), and a second set of 15 fractions was analyzed by 3-h UPLC-ESI-MS/MS separations (45 h total instrument time). CZE-MS/MS produced 70% as many protein IDs (4134 versus 5787) and 60% as many peptide IDs (22 535 versus 36 848) as UPLC-MS/MS with similar instrument time (50 h versus 45 h) but with 50 times smaller total consumed sample amount (1.5 µg versus 75 µg). Surprisingly, CZE generated peaks that were 25% more intense than UPLC for peptides that were identified by both techniques, despite the 50-fold lower loading amount; this high sensitivity reflects the efficient ionization produced by the electrokinetically pumped nanospray interface used in CZE. This report is the first comparison of CZE-MS/MS and UPLC-MS/MS for large-scale eukaryotic proteomic analysis. The numbers of protein and peptide identifications produced by CZE-ESI-MS/MS approach those produced by UPLC-MS/MS, but with nearly two orders of magnitude lower sample amounts.

Coupling Capillary Zone Electrophoresis to a Q Exactive HF Mass Spectrometer for Top-down Proteomics: 580 Proteoform Identifications from Yeast.

We used reversed-phase liquid chromatography to separate the yeast proteome into 23 fractions. These fractions were then analyzed using capillary zone electrophoresis (CZE) coupled to a Q-Exactive HF mass spectrometer using an electrokinetically pumped sheath flow interface. The parameters of the mass spectrometer were first optimized for top-down proteomics using a mixture of seven model proteins; we observed that intact protein mode with a trapping pressure of 0.2 and normalized collision energy of 20% produced the highest intact protein signals and most protein identifications. Then, we applied the optimized parameters for analysis of the fractionated yeast proteome. From this, 580 proteoforms and 180 protein groups were identified via database searching of the MS/MS spectra. This number of proteoform identifications is two times larger than that of previous CZE-MS/MS studies. An additional 3,243 protein species were detected based on the parent ion spectra. Post-translational modifications including N-terminal acetylation, signal peptide removal, and oxidation were identified.

Dynamic pH junction preconcentration in capillary electrophoresis- electrospray ionization-mass spectrometry for proteomics analysis.

Capillary zone electrophoresis (CZE)-electrospray ionization (ESI)-mass spectrometry (MS) is an interesting complimentary technique to reversed phase liquid chromatography (RPLC)-ESI-MS for proteomics research. However, the low sample loading capacity of CZE (typically a few nL) can limit its application for large-scale proteomics. A number of on-line sample preconcentration methods have been developed to increase sample loading volumes. This review considers the dynamic pH junction as a simple on-line sample preconcentration method; this method is well suited for amphiprotic analytes. In the pH junction, these analytes are suspended in a basic buffer, injected by pressure into the capillary, and separated in an acidic background electrolyte, with no changes in either CZE-MS operations or instrumentation. We have demonstrated that the dynamic pH junction method can improve the sample loading volume to sub-µL volumes without significant loss of separation capacity for bottom-up proteomic analysis. The dynamic pH junction based CZE-ESI-MS system has been applied for a number of complex biological samples, including the E. coli proteome, impurities in recombinant antibody therapeutics, and the characterization of the phosphoproteome from a human cell line.

Single Cell Proteomics Using Frog (Xenopus laevis) Blastomeres Isolated from Early Stage Embryos, Which Form a Geometric Progression in Protein Content.

Single cell analysis is required to understand cellular heterogeneity in biological systems. We propose that single cells (blastomeres) isolated from early stage invertebrate, amphibian, or fish embryos are ideal model systems for the development of technologies for single cell analysis. For these embryos, although cell cleavage is not exactly symmetric, the content per blastomere decreases roughly by half with each cell division, creating a geometric progression in cellular content. This progression forms a ladder of single-cell targets for the development of successively higher sensitivity instruments. In this manuscript, we performed bottom-up proteomics on single blastomeres isolated by microdissection from 2-, 4-, 8-, 16-, 32-, and 50-cell Xenopus laevis (African clawed frog) embryos. Over 1 400 protein groups were identified in single-run reversed-phase liquid chromatography-electrospray ionization-tandem mass spectrometry from single balstomeres isolated from a 16-cell embryo. When the mass of yolk-free proteins in single blastomeres decreased from ∼0.8 µg (16-cell embryo) to ∼0.2 µg (50-cell embryo), the number of protein group identifications declined from 1 466 to 644. Around 800 protein groups were quantified across four blastomeres isolated from a 16-cell embryo. By comparing the protein expression among different blastomeres, we observed that the blastomere-to-blastomere heterogeneity in 8-, 16-, 32-, and 50-cell embryos increases with development stage, presumably due to cellular differentiation. These results suggest that comprehensive quantitative proteomics on single blastomeres isolated from these early stage embryos can provide valuable insights into cellular differentiation and organ development.

Quantitative analysis of the supernatant from host and transfected CHO cells using iTRAQ 8-plex technique.

We employed UPLC-MS/MS with iTRAQ 8-plex labeling to quantitatively analyze the supernatant produced by two Chinese hamster ovary (CHO) cell lines (CHO K1SV and CHO CAT-S). In each case, the supernatant from the host and three transfected clones were analyzed at days 5, 7, and 10 of culture. A total of eight iTRAQ 8-plex experiments were performed. For each cell line, the overlap of supernatant protein identifications between transfected clones is over 60%. Over 70% of the supernatant proteins in the CHO K1SV host cell line are present in the CHO CAT-S cell line. For the CHO K1SV cell line, the overlap in supernatant protein identifications between the host cell line and the transfected clones is >59%. For the CHO CAT-S cell line, the overlap between supernatant protein identifications for the transfected clone and host cell is >45%. These differences in the supernatant protein identifications between transfected clones in each cell line and between the two host cell lines are not significant. We used cluster analysis to characterize the change in supernatant protein expression as a function of cell culture time. Roughly <60% of the supernatant proteins show significant change across the three time points (ratio >1.3 or <0.7). We also used cluster analysis to compare changes in supernatant protein expression between the host and three transfected clones at each time point. Greater than 65% of the common proteins in the CHO K1SV cell line supernatant and over 54% in the CHO CAT-S cell line supernatant show no significant expression difference between host and the three transfected clones. Data are available via ProteomeXchange with identifier PXD003462. Biotechnol. Bioeng. 2016;113: 2140-2148. © 2016 Wiley Periodicals, Inc.

Capillary zone electrophoresis for bottom-up analysis of complex proteomes.

Capillary zone electrophoresis (CZE) is emerging as a useful tool in proteomic analysis. Interest arises from dramatic improvements in performance that result from improvements in the background electrolyte used for the separation, the incorporation of advanced sample injection methods, the development of robust and sensitive electrospray interfaces, and the coupling with Orbitrap mass spectrometers with high resolution and sensitivity. The combination of these technologies produces performance that is rapidly approaching the performance of UPLC-based methods for microgram samples and exceeds the performance of UPLC-based methods for mid- to low nanogram samples. These systems now produce over 10 000 peptide IDs in a single 100-min analysis of the HeLa proteome.

Nearly 1000 Protein Identifications from 50 ng of Xenopus laevis Zygote Homogenate Using Online Sample Preparation on a Strong Cation Exchange Monolith Based Microreactor Coupled with Capillary Zone Electrophoresis.

A sulfonate-silica hybrid strong cation exchange monolith microreactor was synthesized and coupled to a linear polyacrylamide coated capillary for online sample preparation and capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS) bottom-up proteomic analysis. The protein sample was loaded onto the microreactor in an acidic buffer. After online reduction, alkylation, and digestion with trypsin, the digests were eluted with 200 mM ammonium bicarbonate at pH 8.2 for CZE-MS/MS analysis using 1 M acetic acid as the background electrolyte. This combination of basic elution and acidic background electrolytes results in both sample stacking and formation of a dynamic pH junction. 369 protein groups and 1274 peptides were identified from 50 ng of Xenopus laevis zygote homogenate, which is comparable with an offline sample preparation method, but the time required for sample preparation was decreased from over 24 h to less than 40 min. Dramatically improved performance was produced by coupling the reactor to a longer separation capillary (∼100 cm) and a Q Exactive HF mass spectrometer. 975 protein groups and 3749 peptides were identified from 50 ng of Xenopus protein using the online sample preparation method.

Capillary zone electrophoresis tandem mass spectrometry detects low concentration host cell impurities in monoclonal antibodies.

We have evaluated CZE-ESI-MS/MS for detection of trace amounts of host cell protein impurities in recombinant therapeutics. Compared to previously published procedures, we have optimized the buffer pH used in the formation of a pH junction to increase injection volume. We also prepared a 5-point calibration curve by spiking 12 standard proteins into a solution of a human mAb. A custom CZE-MS/MS system was used to analyze the tryptic digest of this mixture without depletion of the antibody. CZE generated a ∼70-min separation window (∼90-min total analysis duration) and ∼300-peak capacity. We also analyzed the sample using ultra-performance LC-MS/MS. CZE-MS/MS generated approximately five times higher base peak intensity and more peptide identifications for low-level spiked proteins. Both methods detected all proteins spiked at ∼100 ppm level with respect to the antibody.

Thermally-initiated free radical polymerization for reproducible production of stable linear polyacrylamide coated capillaries, and their application to proteomic analysis using capillary zone electrophoresis-mass spectrometry.

Proteomic analysis using capillary zone electrophoresis (CZE) typically is performed with linear polyacrylamide (LPA) coated capillaries. These capillaries both minimize the adsorption of peptides and proteins to the inner wall of the capillary and decrease electroosmosis, which increases the separation capacity. LPA coating protocols were first reported by Hjerten in 1985. Conventional LPA production is based on the use of tetramethylethylenediamine (TEMED) to catalyze the free-radical polymerization that couples acrylamide to a capillary wall that has been pretreated with γ-methacryloxypropyltrimethoxysilane. The treated capillary is filled with a mixture of monomer, TEMED, and ammonium persulfate; free radical polymerization forms the LPA coating. Over many years, we have observed significant variation in the electroosmotic properties of commercial LPA coated capillaries both along the capillary length and between lots. We believe this variation is due to differences in the time between initiation of the reaction and the filling of the capillary. Here, we report a simple method for the generation of very stable and reproducible coatings. In this protocol, the monomer mixture and an ammonium persulfate initiator are introduced into the capillary without TEMED initiator. The mixture is stable and does not begin polymerization at room temperature. The filled capillary is then heated in a water bath to initiate polymerization in a well-controlled manner. A mixture of four standard proteins was used to evaluate the coating performance. Compared with commercialized LPA capillaries, our LPA capillaries generate much better separation performance and superior protein peak shape in CZE analysis. We also analyzed an intact antibody (MW 150K) by CZE-MS with the new LPA capillary in triplicate runs. The intact antibody generated a Gaussian-shaped electrophoresis peak with 1.2% relative standard deviation in migration time and 8.5% in base peak intensity. An automated CZE-MS system was used to generate 97 successive separations of a BSA tryptic digest over a 145-h period. Separation efficiency averaged over 100,000 theoretical plates across this period with no systematic variation. The LPA coating protocol had excellent batch-to-batch reproducibility with relative standard deviation in migration time<7%, and in separation window<1%.

Over 2300 phosphorylated peptide identifications with single-shot capillary zone electrophoresis-tandem mass spectrometry in a 100 min separation.

Ultraperformance liquid chromatography (UPLC)-electrospray ionization (ESI)-tandem mass spectrometry (MS/MS) is typically employed for phosphoproteome analysis. Alternatively, capillary zone electrophoresis (CZE)-ESI-MS/MS has great potential for phosphoproteome analysis due to the significantly different migration times of phosphorylated and unphosphorylated forms of peptides. In this work, we systematically compared UPLC-MS/MS and CZE-MS/MS for phosphorylated peptide identifications (IDs) using an enriched phosphoproteome from the MCF-10A cell line. When the sample loading amount of UPLC was 10 times higher than that of CZE (2 µg vs 200 ng), UPLC generated more phosphorylated peptide IDs than CZE (3313 vs 1783). However, when the same sample loading amounts were used for CZE and UPLC (2-200 ng), CZE-MS/MS consistently and significantly outperformed UPLC-MS/MS in terms of phosphorylated peptide and total peptide IDs. This superior performance is most likely due to the higher peptide intensity generated by CZE-MS/MS. More importantly, compared with UPLC data from a 2 µg sample, CZE-MS/MS can identify over 500 unique phosphorylated peptides from a 200 ng sample, suggesting that CZE and UPLC are complementary for phosphorylated peptide IDs. With further improved loading capacity via a dynamic pH junction method, 2313 phosphorylated peptides were identified with single-shot CZE-MS/MS in a 100 min analysis. This number of phosphorylated peptide IDs is over 1 order of magnitude higher than the number of phosphorylated peptide IDs previously reported by single-shot CZE-MS/MS.

Integrated strong cation-exchange hybrid monolith coupled with capillary zone electrophoresis and simultaneous dynamic pH junction for large-volume proteomic analysis by mass spectrometry.

A sulfonate-silica hybrid strong cation-exchange (SCX) monolith was synthesized at the proximal end of a capillary zone electrophoresis column and used for on-line solid-phase extraction (SPE) sample preconcentration. Sample was prepared in an acidic buffer and deposited onto the SCX-SPE monolith and eluted using a basic buffer. Electrophoresis was performed in an acidic buffer. This combination of buffers results in formation of a dynamic pH junction, which allows use of relatively large elution buffer volume while maintaining peak efficiency and resolution. All experiments were performed with a 50 µm ID capillary, a 1cm long SCX-SPE monolith, a 60cm long separation capillary, and a electrokinetically pumped nanospray interface. The volume of the capillary is 1.1 µL. By loading 21 µL of a 1×10(-7) M angiotensin II solution, an enrichment factor of 3000 compared to standard electrokinetic injection was achieved on this platform while retaining efficient electrophoretic performance (N=44,000 plates). The loading capacity of the sulfonate SCX hybrid monolith was determined to be ~15 pmol by frontal analysis with 10(-5) M angiotensin II. The system was also applied to the analysis of a 10(-4) mg/mL bovine serum albumin tryptic digest; the protein coverage was 12% and 11 peptides were identified. Finally, by loading 5.5 µL of a 10(-3) mg/mL E. coli digest, 109 proteins and 271 peptides were identified in a 20 min separation; the median separation efficiency generated by these peptides was 25,000 theoretical plates.