scholarly journals Screening a Resource of Recombinant Protein Fragments for Targeted Proteomics

2018 ◽  
Author(s):  
Fredrik Edfors ◽  
Björn Forsström ◽  
Helian Vunk ◽  
David Kotol ◽  
Claudia Fredolini ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Human Plasma ◽  
Recombinant Proteins ◽  
Time Course ◽  
Targeted Proteomics ◽  
Protein Coding ◽  
Protein Fragments ◽  
Peptide Formation ◽  
Digestion Kinetics ◽  
Different Levels

AbstractThe availability of proteomics resources hosting protein and peptide standards, as well as the data describing their analytical performances, will continue to enhance our current capabilities to develop targeted proteomics methods for quantitative biology. This study describes the analysis of a resource of 26,840 individually purified recombinant protein fragments corresponding to more than 16,000 human protein-coding genes. The resource was screened to identify proteotypic peptides suitable for targeted proteomics efforts and we report LC-MS/MS assay coordinates for more than 25,000 proteotypic peptides, corresponding to more than 10,000 unique proteins. Additionally, peptide formation and digestion kinetics were, for a subset of the standards, monitored using a time-course protocol involving parallel digestion of isotope-labelled recombinant protein standards and endogenous human plasma proteins. We show that the strategy by adding isotope-labelled recombinant proteins prior to trypsin digestion enables short digestion protocols (≤60 min) with robust quantitative precision. In a proof-of-concept study, we quantified 23 proteins in human plasma using assay parameters defined in our study and used the standards to describe distinct clusters of individuals linked to different levels of LPA, APOE, SERPINA5 and TFRC. In summary, we describe the use and utility of a resource of recombinant proteins to identify proteotypic peptides useful for targeted proteomics assay development.

scholarly journals A Recombinant Protein Biomarker DDA Library Increases DIA Coverage of Low Abundance Plasma Proteins

2020 ◽  
Author(s):  
Seong Beom Ahn ◽  
Karthik S. Kamath ◽  
Abidali Mohamedali ◽  
Zainab Noor ◽  
Jemma X. Wu ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Human Plasma ◽  
Recombinant Proteins ◽  
Plasma Proteins ◽  
Biomarker Discovery ◽  
Cancer Biomarkers ◽  
Human Blood Plasma ◽  
Spectral Library ◽  
Protein Inference ◽  
High Stringency

AbstractCredible detection and quantification of low abundance proteins from human blood plasma is a major challenge in precision medicine biomarker discovery when using mass spectrometry (MS). Here, we employed a mixture of recombinant proteins in DDA libraries to subsequently detect cancer-associated low abundance plasma proteins using SWATH/DIA. The exemplar DDA recombinant protein spectral library (rPSL) was derived from tryptic digestion of 36 human recombinant proteins that had been previously implicated as possible cancer biomarkers in both our own and other studies. The rPSL was then used to identify proteins from non-depleted colorectal cancer (CRC) plasmas by SWATH-MS. Most (32/36) of the proteins in the rPSL were reliably identified in plasma samples, including 8 proteins (BTC, CXCL10, IL1B, IL6, ITGB6, TGFα, TNF, TP53) not previously detected using high-stringency MS in human plasmas according to PeptideAtlas. The rPSL SWATH-MS protocol was compared to DDA-MS using MARS-depleted and post-digestion peptide fractionated plasmas (here referred to as a human plasma DDA library). Of the 32 proteins identified using rPSL SWATH, only 12 were identified using DDA-MS. The 20 additional proteins exclusively identified by using the rPSL approach with SWATH were mostly lower abundance (i.e., <10ng/ml) plasma proteins. To mitigate FDR concerns, and replicating a more typical approach, the DDA rPSL was also merged into a human plasma DDA library. When SWATH identification was repeated using this merged library, the majority (33/36) of low abundance plasma proteins from the rPSL could still be identified using high-stringency HPP Guidelines v3.0 protein inference criteria.

scholarly journals Screening a Resource of Recombinant Protein Fragments for Targeted Proteomics

2019 ◽  
Vol 18 (7) ◽  
pp. 2706-2718 ◽  
Author(s):  
Fredrik Edfors ◽  
Björn Forsström ◽  
Helian Vunk ◽  
David Kotol ◽  
Claudia Fredolini ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Targeted Proteomics ◽  
Protein Fragments

Functional Properties of p-Anisoylated Plasmin-Staphylokinase Complex

1993 ◽  
Vol 70 (02) ◽  
pp. 326-331 ◽  
Author(s):  
H R Lijnen ◽  
B Van Hoef ◽  
R A G Smith ◽  
D Collen
Keyword(s):  
Human Plasma ◽  
Rate Constant ◽  
Time Course ◽  
Bolus Injection ◽  
Similar Rate ◽  
Clot Lysis ◽  
Lag Phase ◽  
Plasma Clot ◽  
Rapid Clearance ◽  
Stoichiometric Complex

SummaryThe kinetic and fibrinolytic properties of a reversibly acylated stoichiometric complex between human plasmin and recombinant staphylokinase (plasmin-STAR complex) were evaluated. The acylation rate constant of plasmin-STAR by p-amidinophenyl-p’-anisate-HCI was 52 M-1 s-1 and its deacylation rate constant 1.2 × 10-4 s-1 (t½ of 95 min) which are respectively 50-fold and around 3-fold lower than for the plasmin-streptokinase complex. The acylated complex was stable as evidenced by binding to lysine-Sepharose. However, following an initial short lag phase, the acylated plasmin-STAR complex activated plasminogen at a similar rate as the unblocked complex, whereas the acylated plasmin-streptokinase complex did not activate plasminogen. These findings indicate that STAR, unlike streptokinase, dissociates from its acylated complex with plasmin in the presence of excess plasminogen. In agreement with this hypothesis, the time course of the lysis of a 125I-fibrin labeled plasma clot submerged in citrated human plasma, is similar for acylated plasmin-STAR, unblocked plasmin-STAR and free STAR (50% clot lysis in 2 h requires 12 nM of each agent). The plasma clearances of STAR-related antigen following bolus injection in hamsters were 1.0 to 1.5 ml/min for acylated plasmin-STAR, unblocked plasmin-STAR and free STAR, as a result of short initial half-lives of 2.0 to 2.5 min.The dissociation of the anisoylated plasmin-STAR complex and its consequent rapid clearance suggest that it has no apparent advantages as compared to free STAR for clinical thrombolysis.

scholarly journals Multiple Microfermentor Battery: a Versatile Tool for Use with Automated Parallel Cultures of Microorganisms Producing Recombinant Proteins and for Optimization of Cultivation Protocols

2006 ◽  
Vol 72 (8) ◽  
pp. 5225-5231 ◽  
Author(s):  
Emmanuel Frachon ◽  
Vincent Bondet ◽  
Hélène Munier-Lehmann ◽  
Jacques Bellalou
Keyword(s):  
Recombinant Protein ◽  
Recombinant Proteins ◽  
Protein Production ◽  
Batch Cultures ◽  
E Coli ◽  
Working Volume ◽  
Versatile Tool ◽  
Labview Program ◽  
Medium Formulation ◽  
Conventional Medium

ABSTRACT A multiple microfermentor battery was designed for high-throughput recombinant protein production in Escherichia coli. This novel system comprises eight aerated glass reactors with a working volume of 80 ml and a moving external optical sensor for measuring optical densities at 600 nm (OD600) ranging from 0.05 to 100 online. Each reactor can be fitted with miniature probes to monitor temperature, dissolved oxygen (DO), and pH. Independent temperature regulation for each vessel is obtained with heating/cooling Peltier devices. Data from pH, DO, and turbidity sensors are collected on a FieldPoint (National Instruments) I/O interface and are processed and recorded by a LabVIEW program on a personal computer, which enables feedback control of the culture parameters. A high-density medium formulation was designed, which enabled us to grow E. coli to OD600 up to 100 in batch cultures with oxygen-enriched aeration. Accordingly, the biomass and the amount of recombinant protein produced in a 70-ml culture were at least equivalent to the biomass and the amount of recombinant protein obtained in a Fernbach flask with 1 liter of conventional medium. Thus, the microfermentor battery appears to be well suited for automated parallel cultures and process optimization, such as that needed for structural genomics projects.

scholarly journals Different expression levels of two KgmB-His fusion proteins

2005 ◽  
Vol 70 (12) ◽  
pp. 1401-1407 ◽  
Author(s):  
Sandra Markovic ◽  
Sandra Vojnovic ◽  
Milija Jovanovic ◽  
Branka Vasiljevic
Keyword(s):  
Recombinant Proteins ◽  
Fusion Proteins ◽  
Kanamycin Resistance ◽  
Expression Vectors ◽  
E Coli ◽  
C Terminus ◽  
N Terminus ◽  
Different Levels ◽  
Streptomyces Tenebrarius

The KgmB methylase from Streptomyces tenebrarius was expressed and purified using the QIAexpress System. Two expression vectors were made: pQEK-N, which places a (His)6 tag at the N-terminus, and pQEK-C, which places a (His)6 tag at the C-terminus of the recombinant KgmB protein. Kanamycin resistance of the E. coli cells containing either the pQEK-N or the pQEK-C recombinant plasmids confirmed the functionality of both KgmB-His fusion proteins in vivo. Interestingly, different levels of expression were observed between these two recombinant proteins. Namely, KgmB methylase with the (His)6 tag at the N-terminus showed a higher level of expression. Purification of the (His)6-tagged proteins using Ni-NTA affinity chromatography was performed under native conditions and the KgmB methylase with (His)6 tag at the N-terminus was purified to homogeneity >95 %. The recombinant KgmB protein was detected on a Western blot using anti-Sgm antibodies.

scholarly journals Targeted proteomics analysis of plasma proteins using recombinant protein standards for addition only workflows

BioTechniques ◽  
2021 ◽  
Author(s):  
David Kotol ◽  
Andreas Hober ◽  
Linnéa Strandberg ◽  
Anne-Sophie Svensson ◽  
Mathias Uhlén ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Plasma Proteins ◽  
Mass Spectrometry Analysis ◽  
Targeted Proteomics ◽  
Plasma Samples ◽  
Blood Proteins ◽  
Proteomics Analysis ◽  
Protein Targets ◽  
Spectrometry Analysis ◽  
Absolute Concentrations

Targeted proteomics is an attractive approach for the analysis of blood proteins. Here, we describe a novel analytical platform based on isotope-labeled recombinant protein standards stored in a chaotropic agent and subsequently dried down to allow storage at ambient temperature. This enables a straightforward protocol suitable for robotic workstations. Plasma samples to be analyzed are simply added to the dried pellet followed by enzymatic treatment and mass spectrometry analysis. Here, we show that this approach can be used to precisely (coefficient of variation <10%) determine the absolute concentrations in human plasma of hundred clinically relevant protein targets, spanning four orders of magnitude, using simultaneous analysis of 292 peptides. The use of this next-generation analytical platform for high-throughput clinical proteome profiling is discussed.

Longitudinal Plasma Protein Profiling Using Targeted Proteomics and Recombinant Protein Standards

2020 ◽  
Vol 19 (12) ◽  
pp. 4815-4825
Author(s):  
David Kotol ◽  
Helian Hunt ◽  
Andreas Hober ◽  
Max J. Karlsson ◽  
Björn Forsström ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Plasma Protein ◽  
Protein Profiling ◽  
Targeted Proteomics ◽  
Longitudinal Plasma

Abstract 5686: Long RNA sequencing of human plasma exosomes reveals full coverage of diverse protein coding and long non coding RNA

2017 ◽  
Author(s):  
Sudipto K. Chakrabortty ◽  
Lisa Bedford ◽  
Hidefumi Uchiyama ◽  
Vasisht Tadigotla ◽  
Michael D. Valentino ◽  
...  
Keyword(s):  
Human Plasma ◽  
Rna Sequencing ◽  
Protein Coding ◽  
Full Coverage ◽  
Non Coding Rna ◽  
Long Non Coding Rna

SCREENING OF CHLOROPLAST PROMOTERS FOR HEVEA BRASILIENSIS CHLOROPLAST TRANSFORMATION

2015 ◽  
Vol 77 (24) ◽  
Author(s):  
Anas Akmal Ag. Ismail ◽  
Zaima Azira Zainal Abidin ◽  
Zarina Zainuddin
Keyword(s):  
Recombinant Protein ◽  
Plant Species ◽  
Recombinant Proteins ◽  
Hevea Brasiliensis ◽  
Agronomic Traits ◽  
Chloroplast Transformation ◽  
Foreign Protein ◽  
Protein Accumulation ◽  
Nuclear Transformation ◽  
Single Chain

In recent years, the growth in the use of recombinant proteins has grown tremendously. With the aid of the advances in DNA recombinant biotechnology, molecular farming in plants has been applied to meet this increasing demand where plants have emerged as one of the most promising general production platforms for recombinant proteins. Hevea brasiliensis is one of the main commodities in Malaysia and widely cultivated species for commercial production of latex. This important plant has been used to express recombinant proteins such as a single-chain variable fragment (scFv) antibody against the coat protein of Streptococcus gordonii (an oral dental bacterium), human serum albumin and human atrial natriuretic. The genes that encodes for the recombinant proteins were targeted into the nucleus genome of Hevea but the proteins were expressed in low concentration. Generating transgenic plant using chloroplast transformation offers many advantages in comparison to nuclear transformation and many researches have been made to apply this strategy to enhance agronomic traits or produce recombinant protein in several plant species. Since chloroplast is highly polyploidy, it allows high-level foreign protein expression. Given the generally very high foreign protein accumulation rates that can be achieved in transgenic chloroplasts, the aim of this study is to screen a number of chosen endogenous Hevea chloroplast promoters to drive the expression of the reporter gene, uidA for Hevea specific chloroplast transformation vector. Three promoters were chosen for this experiment which are; rbcL, psbA and rrn16 promoters. The putative regions of these promoters were derived from the chloroplast genome sequence of Hevea. Analyses of the three putative promoter regions using multiple sequence alignment with comparable regions from other plant species show significant sequence homology. Further analyses of the putative regions using in-vitro transcription are planned for future study. It is hoped that with the development of an optimized expression vector will allow high expression of valuable recombinant protein in the chloroplast of Hevea.

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