Publications by authors named "Jingnan Huang"

14 Publications

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Assessment of a complete and classified platelet proteome from genome-wide transcripts of human platelets and megakaryocytes covering platelet functions.

Sci Rep 2021 Jun 11;11(1):12358. Epub 2021 Jun 11.

Department of Biochemistry, CARIM, Maastricht University, P.O. Box 616, 6200 MD, Maastricht, The Netherlands.

Novel platelet and megakaryocyte transcriptome analysis allows prediction of the full or theoretical proteome of a representative human platelet. Here, we integrated the established platelet proteomes from six cohorts of healthy subjects, encompassing 5.2 k proteins, with two novel genome-wide transcriptomes (57.8 k mRNAs). For 14.8 k protein-coding transcripts, we assigned the proteins to 21 UniProt-based classes, based on their preferential intracellular localization and presumed function. This classified transcriptome-proteome profile of platelets revealed: (i) Absence of 37.2 k genome-wide transcripts. (ii) High quantitative similarity of platelet and megakaryocyte transcriptomes (R = 0.75) for 14.8 k protein-coding genes, but not for 3.8 k RNA genes or 1.9 k pseudogenes (R = 0.43-0.54), suggesting redistribution of mRNAs upon platelet shedding from megakaryocytes. (iii) Copy numbers of 3.5 k proteins that were restricted in size by the corresponding transcript levels (iv) Near complete coverage of identified proteins in the relevant transcriptome (log2fpkm > 0.20) except for plasma-derived secretory proteins, pointing to adhesion and uptake of such proteins. (v) Underrepresentation in the identified proteome of nuclear-related, membrane and signaling proteins, as well proteins with low-level transcripts. We then constructed a prediction model, based on protein function, transcript level and (peri)nuclear localization, and calculated the achievable proteome at ~ 10 k proteins. Model validation identified 1.0 k additional proteins in the predicted classes. Network and database analysis revealed the presence of 2.4 k proteins with a possible role in thrombosis and hemostasis, and 138 proteins linked to platelet-related disorders. This genome-wide platelet transcriptome and (non)identified proteome database thus provides a scaffold for discovering the roles of unknown platelet proteins in health and disease.
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http://dx.doi.org/10.1038/s41598-021-91661-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8196183PMC
June 2021

Nucleotide-Binding Leucine-Rich Repeat Genes and Are Positive Modulators in the Defense Response to .

Int J Mol Sci 2021 Apr 13;22(8). Epub 2021 Apr 13.

College of Bioscience and Biotechnology, Shenyang Agricultural University, Shenyang 110866, China.

Cucumber powdery mildew caused by is a leaf disease that seriously affects cucumber's yield and quality. This study aimed to report two nucleotide-binding site-leucine-rich repeats (NBS-LRR) genes and , which participated in regulating the resistance of cucumber to . The subcellular localization showed that the CsRSF1 protein was localized in the nucleus, cytoplasm, and cell membrane, while the CsRSF2 protein was localized in the cell membrane and cytoplasm. In addition, the transcript levels of and were different between resistant and susceptible cultivars after treatment with exogenous substances, such as abscisic acid (ABA), methyl jasmonate (MeJA), salicylic acid (SA), ethephon (ETH), gibberellin (GA) and hydrogen peroxide (HO). The expression analysis showed that the transcript levels of and were correlated with plant defense response against . Moreover, the silencing of and impaired host resistance to , but and overexpression improved resistance to in cucumber. These results showed that and genes positively contributed to the resistance of cucumber to . At the same time, and genes could also regulate the expression of defense-related genes. The findings of this study might help enhance the resistance of cucumber to .
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http://dx.doi.org/10.3390/ijms22083986DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8069588PMC
April 2021

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

Anal Chem 2021 01 8;93(2):697-703. Epub 2020 Dec 8.

State Key Laboratory of Genetic Engineering, Department of Biochemistry, School of Life Sciences, Fudan University, Shanghai 200438, China.

Carbodiimide-catalyzed carboxyl and amine conjugation (amidation) has been widely used to protect carboxyl groups. -(3-(Dimethylamino)propyl)-'-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.
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http://dx.doi.org/10.1021/acs.analchem.0c03487DOI Listing
January 2021

Nonredundant Roles of Platelet Glycoprotein VI and Integrin αIIbβ3 in Fibrin-Mediated Microthrombus Formation.

Arterioscler Thromb Vasc Biol 2021 02 3;41(2):e97-e111. Epub 2020 Dec 3.

Department of Biochemistry, CARIM, Maastricht University, the Netherlands (G.P., J.H., I.P., F.S., P.E.J.v.d.M., R.A.S.A., S.P.W., J.W.M.H.).

Objective: Fibrin is considered to strengthen thrombus formation via integrin αIIbβ3, but recent findings indicate that fibrin can also act as ligand for platelet glycoprotein VI. Approach and Results: To investigate the thrombus-forming potential of fibrin and the roles of platelet receptors herein, we generated a range of immobilized fibrin surfaces, some of which were cross-linked with factor XIIIa and contained VWF-BP (von Willebrand factor-binding peptide). Multicolor microfluidics assays with whole-blood flowed at high shear rate (1000 s) indicated that the fibrin surfaces, regardless of the presence of factor XIIIa or VWF-BP, supported platelet adhesion and activation (P-selectin expression), but only microthrombi were formed consisting of bilayers of platelets. Fibrinogen surfaces produced similar microthrombi. Markedly, tiggering of coagulation with tissue factor or blocking of thrombin no more than moderately affected the fibrin-induced microthrombus formation. Absence of αIIbβ3 in Glanzmann thrombasthenia annulled platelet adhesion. Blocking of glycoprotein VI with Fab 9O12 substantially, but incompletely reduced platelet secretion, Ca signaling and aggregation, while inhibition of Syk further reduced these responses. In platelet suspension, glycoprotein VI blockage or Syk inhibition prevented fibrin-induced platelet aggregation. Microthrombi on fibrin surfaces triggered only minimal thrombin generation, in spite of thrombin binding to the fibrin fibers.

Conclusions: Together, these results indicate that fibrin fibers, regardless of their way of formation, act as a consolidating surface in microthrombus formation via nonredundant roles of platelet glycoprotein VI and integrin αIIbβ3 through signaling via Syk and low-level Ca rises.
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http://dx.doi.org/10.1161/ATVBAHA.120.314641DOI Listing
February 2021

Impaired iloprost-induced platelet inhibition and phosphoproteome changes in patients with confirmed pseudohypoparathyroidism type Ia, linked to genetic mutations in GNAS.

Sci Rep 2020 07 9;10(1):11389. Epub 2020 Jul 9.

Department of Biochemistry, CARIM, Maastricht University, PO Box 616, 6200 MD, Maastricht, The Netherlands.

Patients diagnosed with pseudohypoparathyroidism type Ia (PHP Ia) suffer from hormonal resistance and abnormal postural features, in a condition classified as Albright hereditary osteodystrophy (AHO) syndrome. This syndrome is linked to a maternally inherited mutation in the GNAS complex locus, encoding for the GTPase subunit Gsα. Here, we investigated how platelet phenotype and omics analysis can assist in the often difficult diagnosis. By coupling to the IP receptor, Gsα induces platelet inhibition via adenylyl cyclase and cAMP-dependent protein kinase A (PKA). In platelets from seven patients with suspected AHO, one of the largest cohorts examined, we studied the PKA-induced phenotypic changes. Five patients with a confirmed GNAS mutation, displayed impairments in Gsα-dependent VASP phosphorylation, aggregation, and microfluidic thrombus formation. Analysis of the platelet phosphoproteome revealed 2,516 phosphorylation sites, of which 453 were regulated by Gsα-PKA. Common changes in the patients were: (1) a joint panel of upregulated and downregulated phosphopeptides; (2) overall PKA dependency of the upregulated phosphopeptides; (3) links to key platelet function pathways. In one patient with GNAS mutation, diagnosed as non-AHO, the changes in platelet phosphoproteome were reversed. This combined approach thus revealed multiple phenotypic and molecular biomarkers to assist in the diagnosis of suspected PHP Ia.
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http://dx.doi.org/10.1038/s41598-020-68379-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7347634PMC
July 2020

Thrombo-Inflammation in Cardiovascular Disease: An Expert Consensus Document from the Third Maastricht Consensus Conference on Thrombosis.

Thromb Haemost 2020 Apr 14;120(4):538-564. Epub 2020 Apr 14.

Department of Hematology and Central Hematology Laboratory, Inselspital, Bern University Hospital, University of Bern, Bern, Switzerland; Center for Thrombosis and Hemostasis, University Medical Center of the Johannes Gutenberg University, Mainz, Germany; Haemostasis Research Unit, University College London, London, United Kingdom.

Thrombo-inflammation describes the complex interplay between blood coagulation and inflammation that plays a critical role in cardiovascular diseases. The third Maastricht Consensus Conference on Thrombosis assembled basic, translational, and clinical scientists to discuss the origin and potential consequences of thrombo-inflammation in the etiology, diagnostics, and management of patients with cardiovascular disease, including myocardial infarction, stroke, and peripheral artery disease. This article presents a state-of-the-art reflection of expert opinions and consensus recommendations regarding the following topics: (1) challenges of the endothelial cell barrier; (2) circulating cells and thrombo-inflammation, focused on platelets, neutrophils, and neutrophil extracellular traps; (3) procoagulant mechanisms; (4) arterial vascular changes in atherogenesis; attenuating atherosclerosis and ischemia/reperfusion injury; (5) management of patients with arterial vascular disease; and (6) pathogenesis of venous thrombosis and late consequences of venous thromboembolism.
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http://dx.doi.org/10.1055/s-0040-1708035DOI Listing
April 2020

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

Anal Chem 2020 04 9;92(7):4742-4748. Epub 2020 Mar 9.

State Key Laboratory of Genetic Engineering, Department of Biochemistry, School of Life Sciences, Fudan University, Shanghai 200438, China.

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 sic trong ation echange 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, , 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.
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http://dx.doi.org/10.1021/acs.analchem.9b05280DOI Listing
April 2020

Brain transcriptome of honey bees (Apis mellifera) exhibiting impaired olfactory learning induced by a sublethal dose of imidacloprid.

Pestic Biochem Physiol 2019 May 4;156:36-43. Epub 2019 Feb 4.

College of Bee Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China. Electronic address:

Declines in honey bee populations represent a worldwide concern. The widespread use of neonicotinoid insecticides has been one of the factors linked to these declines. Sublethal doses of a neonicotinoid insecticide, imidacloprid, has been reported to cause olfactory learning deficits in honey bees via impairment of the target organ, the brain. In the present study, olfactory learning of honey bees was compared between controls and imidacloprid-treated bees. The brains of imidacloprid-treated and control bees were used for comparative transcriptome analysis by RNA-Seq to elucidate the effects of imidacloprid on honey bee learning capacity. The results showed that the learning performance of imidacloprid-treated bees was significantly impaired in comparison with control bees after chronic oral exposure to imidacloprid (0.02 ng/μl) for 11 days. Gene expression profiles between imidacloprid treatment and the control revealed that 131 genes were differentially expressed, of which 130 were downregulated in imidacloprid-treated bees. Validation of the RNA-Seq data using qRT-PCR showed that the results of qRT-PCR and RNA-Seq exhibited a high level of agreement. Gene ontology annotation indicated that the oxidation-reduction imbalance might exist in the brain of honey bees due to oxidative stress induced by imidacloprid exposure. KEGG and ingenuity pathway analysis revealed that transient receptor potential and Arrestin 2 in the phototransduction pathway were significantly downregulated in imidacloprid-treated bees, and that five downregulated genes have causal effects on behavioral response inhibition in imidacloprid-treated bees. Our results suggest that downregulation of brain genes involved in immune, detoxification and chemosensory responses may result in decreased olfactory learning capabilities in imidacloprid-treated bees.
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http://dx.doi.org/10.1016/j.pestbp.2019.02.001DOI Listing
May 2019

Lys-C/Arg-C, a More Specific and Efficient Digestion Approach for Proteomics Studies.

Anal Chem 2018 08 1;90(16):9700-9707. Epub 2018 Aug 1.

State Key Laboratory of Genetic Engineering, Department of Biochemistry, School of Life Sciences , Fudan University , Shanghai 200438 , China.

Nowadays, bottom-up approaches are predominantly adopted in proteomics studies, which necessitate a proteolysis step prior to MS analysis. Trypsin is often the best protease in choice due to its high specificity and MS-favored proteolytic products. A lot of efforts have been made to develop a superior digestion approach but hardly succeed, especially in large-scale proteomics studies. Herein, we report a new tandem digestion using Lys-C and Arg-C, termed Lys-C/Arg-C, which has been proven to be more specific and efficient than trypsin digestion. Reanalysis of our previous data ( Anal. Chem. 2018, 90 (3), 1554-1559) revealed that both Lys-C and Arg-C are trypsin-like proteases and perform better when considered as trypsin. In particular, for Arg-C, the identification capacity is increased to 2.6 times and even comparable with trypsin. The good complementarity, high digestion efficiency, and high specificity of Lys-C and Arg-C prompt the Lys-C/Arg-C digestion. We systematically evaluated Lys-C/Arg-C digestion using qualitative and quantitative proteomics approaches and confirmed its superior performance in digestion specificity, efficiency, and identification capacity to the currently widely used trypsin and Lys-C/trypsin digestions. As a result, we concluded that the Lys-C/Arg-C digestion approach would be the choice of next-generation digestion approach in both qualitative and quantitative proteomics studies. Data are available via ProteomeXchange with identifier PXD009797.
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http://dx.doi.org/10.1021/acs.analchem.8b02448DOI Listing
August 2018

Morphological and Near-Field Properties of Silver Columnar Thin Film for Surface-Enhanced Raman Scattering.

J Nanosci Nanotechnol 2018 Apr;18(4):2803-2810

State Key Laboratory of Optoelectronic Materials and Technologies, Guangzhou, 510275, People's Republic of China; School of Physics and Engineering, Sun Yat-Sen University Guangzhou, 510275, People's Republic of China.

Noble metal sculptured thin films have attracted great research interest last decade as competitive surface-enhanced Raman scattering (SERS) substrates. However, the influences of the deposition conditions and the morphology on the plasmonic properties and SERS performance of the metal sculptured thin films have not been well understood due to the complexities of the morphology. In this work, the influences of the deposition angle and the height are investigated in both experiment and numerical simulation. A more accurate geometrical model based on the binarized scanning electron microscope images has been utilized to study the near-field plasmonic properties of Ag column thin films by taking account of the geometry irregularities, size distributions and random arrangement of the columns. It's found that the cross-sectional electric field enhancement is mainly dominated by the column density. When the deposition angle increases from 68° to 82° the SERS enhancement factors increases monotonously due to the increase of the self-shadow effect. While with the increase of height the SERS enhancement factors firstly increase to the largest value of 3.05 × 108 at the thickness of 694 nm then decrease because of competitive growth mechanism during the deposition. The detection limit of the optimized sample is found to be lower than 10-12 M. Our work could be helpful in understanding the SERS mechanism and useful to the optimization of metal sculptured thin films as SERS substrates.
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http://dx.doi.org/10.1166/jnn.2018.14539DOI Listing
April 2018

Enzyme and Chemical Assisted N-Terminal Blocked Peptides Analysis, ENCHANT, as a Selective Proteomics Approach Complementary to Conventional Shotgun Approach.

J Proteome Res 2018 01 7;17(1):212-221. Epub 2017 Dec 7.

State Key Laboratory of Genetic Engineering, Department of Biochemistry, School of Life Sciences, Fudan University , Shanghai 200438, China.

Shotgun (bottom-up) approach has been widely applied in large-scale proteomics studies. The inherent shortages of shotgun approach lie in that the generated peptides often overwhelm the analytical capacity of current LC-MS/MS systems and that high-abundance proteins often hamper the identification of low-abundance proteins when analyzing complex samples. To reduce the sample complexity and relieve the problems caused by abundant proteins, herein we introduce a modified selective proteomics approach, termed ENCHANT, for enzyme and chemical assisted N-terminal blocked peptides analysis. Modified from our previous Nα-acetylome approach, ENCHANT aims to analyze three kinds of peptides, acetylated protein N-termini, N-terminal glutamine and N-terminal cysteine containing peptides. Application of ENCHANT to HeLa cells allowed to identify 3375 proteins, 19.6% more than that by conventional shotgun approach. More importantly, ENCHANT demonstrated an excellent complementarity to conventional shotgun approach with the overlap of 34.5%. In terms of quantification using data independent acquisition (DIA) technology, ENCHANT quantified 23.9% more proteins than conventional shotgun approach with the overlap of 27.6%. Therefore, our results strongly suggest that ENCHANT is a promising selective proteomics approach, which is complementary to conventional shotgun approach in both qualitative and quantitative proteomics studies. Data are available via ProteomeXchange with identifier PXD007863.
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http://dx.doi.org/10.1021/acs.jproteome.7b00521DOI Listing
January 2018

An Approach to Incorporate Multi-Enzyme Digestion into C-TAILS for C-Terminomics Studies.

Proteomics 2018 01 14;18(1). Epub 2017 Dec 14.

State Key Laboratory of Genetic Engineering, Department of Biochemistry, School of Life Sciences, Fudan University, Shanghai, China.

Protein C-termini study is still a challenging task and far behind its counterpart, N-termini study. MS based C-terminomics study is often hampered by the low ionization efficiency of C-terminal peptides and the lack of efficient enrichment methods. We previously optimized the C-terminal amine-based isotope labeling of substrates (C-TAILS) method and identified 369 genuine protein C-termini in Escherichia coli. A key limitation of C-TAILS is that the prior protection of amines and carboxylic groups at protein level makes Arg-C as the only specific enzyme in practice. Herein, we report an approach combining multi-enzyme digestion and C-TAILS, which significantly increases the identification rate of C-terminal peptides and consequently improves the applicability of C-TAILS in biological studies. We carry out a systematic study and confirm that the omission of the prior amine protection at protein level has a negligible influence and allows the application of multi-enzyme digestion. We successfully apply five different enzyme digestions to C-TAILS, including trypsin, Arg-C, Lys-C, Lys-N, and Lysarginase. As a result, we identify a total of 722 protein C-termini in E. coli, which is at least 66% more than the results using any single enzyme. Moreover, the favored enzyme and enzyme combination are discovered. Data are available via ProteomeXchange with identifier PXD004275.
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http://dx.doi.org/10.1002/pmic.201700034DOI Listing
January 2018

An optimized guanidination method for large-scale proteomic studies.

Proteomics 2016 07 8;16(13):1837-46. Epub 2016 Jun 8.

State Key Laboratory of Genetic Engineering, Department of Biochemistry, School of Life Sciences, Fudan University, Shanghai, P. R. China.

As an ε-amine specific derivatization method, guanidination is widely used in proteomic studies for mainly two reasons: the significant improvement in ionization efficiency and the selective protection of ε-amine. Herein, we employed a systematic comparison of two widely used guanidination approaches and revealed the advantages and disadvantages of each method. The sodium buffer based approach resulted in an unexpected side modification, +57 Da, which is reported for the first time; whereas the ammonium buffer based approach resulted in relatively lower yield. We carried out an optimization study by testing different buffer compositions, pH, temperatures and reaction times, and consequently discovered the optimized guanidination condition. Furthermore, we decoded the +57 Da side product as the addition of C2 H3 NO and proposed a possible mechanism of the side reaction. Importantly, our study demonstrated that mass spectrometry is a powerful tool in discovering minor side reactions which are often impossible by other techniques, and hence suggested that chemical derivatization methods should be investigated more carefully prior to extensive applications in proteomics field.
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http://dx.doi.org/10.1002/pmic.201500226DOI Listing
July 2016

Systematic Optimization of C-Terminal Amine-Based Isotope Labeling of Substrates Approach for Deep Screening of C-Terminome.

Anal Chem 2015 Oct 1;87(20):10354-61. Epub 2015 Oct 1.

State Key Laboratory of Genetic Engineering, Department of Biochemistry, School of Life Sciences, Fudan University , Shanghai 200438, China.

It is well-known that protein C-termini play important roles in various biological processes, and thus the precise characterization of C-termini is essential for fully elucidating protein structures and understanding protein functions. Although many efforts have been made in the field during the latest 2 decades, the progress is still far behind its counterpart, N-termini, and it necessitates more novel or optimized methods. Herein, we report an optimized C-termini identification approach based on the C-terminal amine-based isotope labeling of substrates (C-TAILS) method. We optimized the amidation reaction conditions to achieve higher yield of fully amidated product. We evaluated different carboxyl and amine blocking reagents and found the superior performance of Ac-NHS and ethanolamine. Replacement of dimethylation with acetylation for Lys blocking resulted in the identification of 232 C-terminal peptides in an Escherichia coli sample, about 42% higher than the conventional C-TAILS. A systematic data analysis revealed that the optimized method is unbiased to the number of lysine in peptides, more reproducible and with higher MASCOT scores. Moreover, the introduction of the Single-Charge Ion Inclusion (SCII) method to alleviate the charge deficiency of small peptides allowed an additional 26% increase in identification number. With the optimized method, we identified 481 C-terminal peptides corresponding to 369 C-termini in E. coli in a triplicate experiments using 80 μg each. Our optimized method would benefit the deep screening of C-terminome and possibly help discover some novel C-terminal modifications. Data are available via ProteomeXchange with identifier PXD002409.
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http://dx.doi.org/10.1021/acs.analchem.5b02451DOI Listing
October 2015
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