scholarly journals Standard Flow Multiplexed Proteomics (SFloMPro)—An Accessible Alternative to NanoFlow Based Shotgun Proteomics

Proteomes ◽  
2022 ◽  
Vol 10 (1) ◽  
pp. 3
Author(s):  
Benjamin C. Orsburn ◽  
Sierra D. Miller ◽  
Conor J. Jenkins

Multiplexed proteomics using isobaric tagging allows for simultaneously comparing the proteomes of multiple samples. In this technique, digested peptides from each sample are labeled with a chemical tag prior to pooling sample for LC-MS/MS with nanoflow chromatography (NanoLC). The isobaric nature of the tag prevents deconvolution of samples until fragmentation liberates the isotopically labeled reporter ions. To ensure efficient peptide labeling, large concentrations of labeling reagents are included in the reagent kits to allow scientists to use high ratios of chemical label per peptide. The increasing speed and sensitivity of mass spectrometers has reduced the peptide concentration required for analysis, leading to most of the label or labeled sample to be discarded. In conjunction, improvements in the speed of sample loading, reliable pump pressure, and stable gradient construction of analytical flow HPLCs has continued to improve the sample delivery process to the mass spectrometer. In this study we describe a method for performing multiplexed proteomics without the use of NanoLC by using offline fractionation of labeled peptides followed by rapid “standard flow” HPLC gradient LC-MS/MS. Standard Flow Multiplexed Proteomics (SFloMPro) enables high coverage quantitative proteomics of up to 16 mammalian samples in about 24 h. In this study, we compare NanoLC and SFloMPro analysis of fractionated samples. Our results demonstrate that comparable data is obtained by injecting 20 µg of labeled peptides per fraction with SFloMPro, compared to 1 µg per fraction with NanoLC. We conclude that, for experiments where protein concentration is not strictly limited, SFloMPro is a competitive approach to traditional NanoLC workflows with improved up-time, reliability and at a lower relative cost per sample.

2020 ◽  
Author(s):  
Conor Jenkins ◽  
Ben Orsburn

Multiplexed proteomics using isobaric tagging allows for simultaneously comparing the proteomes of multiple samples. In this technique, digested peptides from each sample are labeled with a chemical tag prior to pooling sample for LC-MS/MS with nanoflow chromatography (NanoLC). The isobaric nature of the tag prevents deconvolution of samples until fragmentation liberates the isotopically labeled reporter ions. To ensure efficient peptide labeling, large concentrations of labeling reagents are included in the reagent kits to allow scientists to use high ratios of chemical label per peptide. The increasing speed and sensitivity of mass spectrometers has reduced the peptide concentration required for analysis, leading to most of the label or labeled sample to be discarded. In conjunction, improvements in the speed of sample loading, reliable pump pressure, and stable gradient construction of analytical flow HPLCs has continued to improve the sample delivery process to the mass spectrometer. In this study we describe a method for performing multiplexed proteomics without the use of NanoLC by using offline fractionation of labeled peptides followed by rapid standard flow HPLC gradient LC-MS/MS. Standard Flow Multiplexed Proteomics (SFloMPro) enables high coverage quantitative proteomics of up to 16 mammalian samples in about 24 hours. In this study, we compare NanoLC and SFloMPro analysis of fractionated samples. Our results demonstrate that comparable data is obtained by injecting 20 micrograms of labeled peptides per fraction with SFloMPro, compared to 1 microgram per fraction with NanoLC. We conclude that, for experiments where protein concentration is not strictly limited, SFloMPro is a competitive approach to traditional NanoLC workflows with improved up-time, reliability and at a lower relative cost per sample.


GigaScience ◽  
2021 ◽  
Vol 10 (1) ◽  
Author(s):  
Taras K Oleksyk ◽  
Walter W Wolfsberger ◽  
Alexandra M Weber ◽  
Khrystyna Shchubelka ◽  
Olga T Oleksyk ◽  
...  

Abstract Background The main goal of this collaborative effort is to provide genome-wide data for the previously underrepresented population in Eastern Europe, and to provide cross-validation of the data from genome sequences and genotypes of the same individuals acquired by different technologies. We collected 97 genome-grade DNA samples from consented individuals representing major regions of Ukraine that were consented for public data release. BGISEQ-500 sequence data and genotypes by an Illumina GWAS chip were cross-validated on multiple samples and additionally referenced to 1 sample that has been resequenced by Illumina NovaSeq6000 S4 at high coverage. Results The genome data have been searched for genomic variation represented in this population, and a number of variants have been reported: large structural variants, indels, copy number variations, single-nucletide polymorphisms, and microsatellites. To our knowledge, this study provides the largest to-date survey of genetic variation in Ukraine, creating a public reference resource aiming to provide data for medical research in a large understudied population. Conclusions Our results indicate that the genetic diversity of the Ukrainian population is uniquely shaped by evolutionary and demographic forces and cannot be ignored in future genetic and biomedical studies. These data will contribute a wealth of new information bringing forth a wealth of novel, endemic and medically related alleles.


2019 ◽  
Vol 21 (1) ◽  
pp. 179 ◽  
Author(s):  
Ramamurthy Mahalingam

Barley seeds are one of the main ingredients of the malting industry for brewing beer. The barley rootlets that are separated from the kilned seeds at the end of the malting process and used as animal feed are one of the byproducts of this industry. In this study, the proteome of rootlets derived from two stages of the malting process, germination and kilning, from a popular malting barley variety were analyzed. A label-free shotgun proteomics strategy was used to identify more than 800 proteins from the barley rootlets. A high coverage and high confidence Gene Ontology annotations of the barley genome was used to facilitate the functional annotation of the proteins that were identified in the rootlets. An analysis of these proteins using Kellogg Encyclopedia of Genes and Genomes (KEGG) and Plant Reactome databases indicated the enrichment of pathways associated with phytohormones, protein biosynthesis, secondary metabolism, and antioxidants. Increased levels of jasmonic acid and auxin in the rootlets further supported the in silico analysis. As a rich source of proteins and amino acids use of these by-products of the malting industry for animal feed is validated. This study also indicates rootlets as a potential source of naturally occurring phenylpropanoids and antioxidants that can be further exploited in the development of functional foods.


2021 ◽  
Author(s):  
Yuting Yuan ◽  
Benjamin C Orsburn

The introduction of isobaric tagging reagents enabled more accurate, high-throughput quantitative proteomics by enabling samples to be multiplexed. One drawback of these workflows is the relative expense of the proprietary chemical reagents, which is often only second to the expense of the instruments themselves. These highly reactive chemical tags are only commercially available in relatively large aliquots compared to the typical amounts of peptides analyzed in proteomic workflows today. Excess reagents are typically disposed of following labeling of small batches or within a few weeks of opening. We present a simple procedure to aliquot commercial isobaric tagging reagents and demonstrate the successful and high efficiency labeling of multiple samples over a period of six months. The samples presented herein were selected as the most diverse samples labeled by prepared aliquots from a single labeling reagent kit. We observe comparable labeling efficiency from 100 microgram to 100 picograms of peptide when labeling samples from both human digest standards, cancer cell lines prepared in-house and from cells directly obtained from organ donations, despite differences in cell type, lysis and digestion. No labeling experiment of whole human proteomics samples achieved less than 92% labeling efficiency over this period. When preparing phosphoproteomic samples from a cancer cell line digest at approximately 6 months from the date of the aliquoting procedure, we observed a decrease in labeling efficiency to approximately 86%, indicating we are approaching the end of the useful lifetime of these prepared aliquots. Over this period, we have effectively reduced the reagent costs of each labeling experiment to less than 10% of the predicted costs when following the manufacturer instructions for use and disposal. While aliquoting of reagents can be performed by hand, we provide a complete template for automatic aliquoting using an affordable liquid handling robot, including plans for 3D printing of parts we have found useful for streamlining this procedure.


2019 ◽  
Author(s):  
Aimee Rinas ◽  
Conor Jenkins ◽  
Ben Orsburn

Capillary electrophoresis coupled electrospray ionization mass spectrometry (CE-MS) has long existed as a theoretical alternative to liquid chromatography coupled mass spectrometry (LC-MS). Until recently, however, the coupling of these technologies has occupied only a small niche within specific applications. A recent innovation in CE-MS is the ZipChip interface system from 908 devices that was pioneered by the Ramsay lab at NC State. This newly available source offers advantages over previous CE-MS interfaces including both relative ease of use and direct compatibility to thousands of mass spectrometers currently in use throughout the world with no hardware alterations. The ZipChip CE-MS has been demonstrated in recent studies to provide high resolution and rapid separations for the analysis of intact proteins, glycoproteins and glycosylated peptides, with more applications likely on the way. In this study we assess the capabilities of the ZipChip system in the context of high throughput global shotgun proteomics experiments. We find that on a high field Orbitrap system we can repeatedly identify as many as 800 unique protein groups in an experiment using a run time of 12 minutes. We find the ZipChip CE-MS system to be widely applicable for both data dependent and data independent acquisition experiments as well as targeted experiments. We conclude that the ZipChip is an attractive alternative solution to traditional nanoflow ESI-MS/MS for the analysis of the genomes of single celled organisms and for offline fractionation of eukaryotic proteomes.


2021 ◽  
pp. jmedgenet-2021-108160
Author(s):  
Katarzyna Klonowska ◽  
Elizabeth A. Thiele ◽  
Joannes M. Grevelink ◽  
Aaron R. Thorner ◽  
David J. Kwiatkowski

Tuberous sclerosis complex (TSC) is a genetic syndrome due to mutations in either TSC1 or TSC2, leading to the development of hamartomatous tumours at multiple body sites, including facial skin (facial angiofibroma (FAF)), brain (cortical tubers) and kidney (angiomyolipoma (AML)). In this report, we describe an individual with minimal TSC clinical features, who had ‘no mutation identified’ (NMI) by prior genetic testing in a clinical laboratory. Our massively parallel sequencing (MPS) analysis of multiple samples from different body sites and tumours (including blood, saliva, normal skin, AML and FAF) revealed an extraordinary situation in which FAF and AML had completely independent inactivating biallelic variants in TSC2, not present in other matched samples. This suggests that the two different lesions (AML and FAF) are not due to the same underlying germline or mosaic mutation, rather both are likely sporadic events. This case demonstrates the relevance of thorough clinical examination, high-coverage MPS of multiple tumours and matched normal tissues, and appropriate genetic counselling for individuals with marginal TSC features and possible TSC1 or TSC2 mosaicism.


Author(s):  
Y. Taniguchi ◽  
E. Nakazawa ◽  
S. Taya

Imaging energy filters can add new information to electron microscopic images with respect to energy-axis, so-called electron spectroscopic imaging (ESI). Recently, many good results have been reported using this imaging technique. ESI also allows high-contrast observation of unstained biological samples, becoming a trend of the field of morphology. We manufactured a new type of energy filter as a trial production. This energy filter consists of two magnets, and we call γ-filter since the trajectory of electrons shows ‘γ’-shape inside the filter. We evaluated the new energyγ-filter TEM with the γ-filter.Figure 1 shows schematic view of the electron optics of the γ-type energy filter. For the determination of the electron-optics of the γ-type energy filter, we used the TRIO (Third Order Ion Optics) program which has been developed for the design of high resolution mass spectrometers. The TRIO takes the extended fringing fields (EFF) into consideration. EFF makes it difficult to design magnetic energy filters with magnetic sector fields.


Author(s):  
L. J. Brenner ◽  
D. G. Osborne ◽  
B. L. Schumaker

Exposure of the ciliate, Tetrahymena pyriformis, strain WH6, to normal human or rabbit sera or mouse ascites fluids induces the formation of large cytoplasmic bodies. By electron microscopy these (LB) are observed to be membrane-bounded structures, generally spherical and varying in size (Fig. 1), which do not resemble the food vacuoles of cells grown in proteinaceous broth. The possibility exists that the large bodies represent endocytic vacuoles containing material concentrated from the highly nutritive proteins and lipoproteins of the sera or ascites fluids. Tetrahymena mixed with bovine serum albumin or ovalbumin solutions having about the same protein concentration (7g/100 ml) as serum form endocytic vacuoles which bear little resemblance to the serum-induced LB. The albumin-induced structures (Fig. 2) are irregular in shape, rarely spherical, and have contents which vary in density and consistency. In this paper an attempt is made to formulate the sequence of events which might occur in the formation of the albumin-induced vacuoles.


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