Development of a Univariate Membrane-Based Mid-Infrared Method for Protein Quantitation and Total Lipid Content Analysis of Biological Samples

Strug, Ivona; Utzat, Christopher D.; Cappione, Amedeo; Gutierrez, Sara; Amara, Ryan; Lento, Joseph; Capito, Florian; Skudas, Romas; Chernokalskaya, Elena; Nadler, Timothy

doi:10.1155/2014/657079

Cited by 28 publications

(33 citation statements)

References 45 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…However, once both iron and sulfur are provided to the scaffold, as is the case for FeS-fIscU, these data are consistent with the majority of the iron coordinated as a FeS-cluster. 24,25 This results suggest a model where Fe translocates from an initial Fe-binding site to the FeS-cluster assembly site after or concurrent with accepting persulfide sulfur from Nfs1. The pattern of Fe-N, Fe-S, and Fe-Fe scattering in the FeS-fIscU EXAFS is consistent with the expected FeS-cluster coordination at the active site of fIscU, which contains three cysteines and one histidine, although the high coordination number for the Fe-O/N environment could suggest a portion of the metal still exists at the initial metal-binding site.…”

Section: Discussionmentioning

confidence: 76%

See 1 more Smart Citation

In vitro characterization of a novel Isu homologue from Drosophila melanogaster for de novo FeS-cluster formation

et al. 2017

View full text Add to dashboard Cite

FeS-clusters are utilized by numerous proteins within several biological pathways that are essential for life. In eukaryotes, the primary FeS-cluster production pathway is the mitochondrial iron-sulfur cluster (ISC) pathway. In Saccharomyces cerevisiae, de novo FeS-cluster formation is accomplished through coordinated assembly with the substrates iron and sulfur by the scaffold assembly protein “Isu1”. Sulfur for cluster assembly is provided by cysteine desulfurase “Nfs1”, a protein that works in union with its accessory protein “Isd11”. Frataxin “Yfh1” helps direct cluster assembly by serving as a modulator of Nfs1 activity, by assisting in the delivery of sulfur and Fe(II) to Isu1, or more likely through a combination of these and other possible roles. In vitro studies on the yeast ISC machinery have been limited, however, due to the inherent instability of recombinant Isu1. Isu1 is a molecule prone to degradation and aggregation. To circumvent Isu1 instability, we have replaced yeast Isu1 with the fly ortholog to stabilize our in vitro ISC assembly system and assist us in elucidating molecular details of the yeast ISC pathway. Our laboratory previously observed that recombinant frataxin from Drosophila melanogaster has remarkable stability compared to the yeast ortholog. Here we provide the first characterization of D. melanogaster Isu1 (fIscU) and demonstrate its ability to function within the yeast ISC machinery both in vivo and in vitro. Recombinant fIscU has similar physical properties similar to that of yeast Isu1. It functions as a stable dimer with similar Fe(II) affinity and ability to form two 2Fe-2S clusters as the yeast dimer. The fIscU and yeast ISC proteins are compatible in vitro; addition of Yfh1 to Nfs1-Isd11 increases the rate of FeS-cluster formation on fIscU to a similar extent observed with Isu1. Finally, fIscU expressed in mitochondria of a yeast strain lacking Isu1 (and its paralog Isu2) is able to completely reverse the deletion phenotypes. These results demonstrate fIscU can functionally replace yeast Isu1 and it can serve as a powerful tool for exploring molecular details within the yeast ISC pathway.

show abstract

Section: Discussionmentioning

confidence: 76%

“…Purified fractions were pooled and concentrated via Amicon centrifugation. Protein concentration was determined using a Direct-detect IR spectrometer 24 (Millipore) and protein purity was assessed via SDS-PAGE. Typical protein purities were ~95%.…”

Section: Methodsmentioning

confidence: 99%

In vitro characterization of a novel Isu homologue from Drosophila melanogaster for de novo FeS-cluster formation

et al. 2017

View full text Add to dashboard Cite

show abstract

“…To date, purity testing and quantification of peptides and proteins have usually been performed by UV absorption, by middle infrared absorption and by mass spectrometry against an appropriate reference standard . The amide bond is monitored at λ = 214 nm, phenylalanine (Phe) at 260 nm, tyrosine (Tyr) at 278 nm, and tryptophan (Trp) at 280 nm.…”

Section: Introductionmentioning

confidence: 99%

Quantification of hydrolyzed peptides and proteins by amino acid fluorescence

Allenspach¹,

Fuchs²,

Doriot³

et al. 2018

Journal of Peptide Science

View full text Add to dashboard Cite

Reliable quantification of peptides and proteins is essential for drug discovery. We report the successful development and validation of an accurate and broadly applicable high performance liquid chromatography hyphenated to fluorescence detector procedure for the quantitative determination of the aromatic amino acids tyrosine, phenylalanine, and tryptophan, without relying on derivatization chemistry. Using ion-pair chromatography, fluorescent amino acids were clearly separated within 10 minutes. The hydrolysis of peptides was performed under acidic and heated conditions to yield the monomeric building blocks. Various protecting agents were tested to ensure tryptophan stability. The presented analytical method accurately (>95%) quantifies all fluorescent residues. The power of the method was confirmed by correct quantification of protein reference standard to 98.6% over all fluorescence traces. The method allowed us to identify pre-analytical differences between the nominal and actual concentrations of 12 peptide solutions. Salt formation, weighing errors, and other pre-analytical pitfalls resulted in noteworthy differences of up to 85% between the indicated and actual concentration of peptide solutions, subsequently leading to false positive or negative interpretation of activity data. Finally, only one solution is needed to perform quantification as well as UV-purity tests and can further be used as stock solution for activity testing.

show abstract

“…(); 3 Nagy (); 4 Lichtenbelt (); 5 Hwang, Larivière & Messier (); 6 Barthelmess, Phillips & Schuckers (); 7 Reynolds & Kunz (); 8 Zor & Selinger (); 9 Strug et al . (); 10 Vetter & Thurnhofer (); 11 Maleknia & Johnson (); 12 Hill et al . (); 13 Chaytor (); 14 Sterner & Elser (); 15 VanGuilder, Vrana & Freeman (); 16 Schulenburg et al .…”

Section: Introductionmentioning

confidence: 99%

Moving beyond body condition indices as an estimate of fitness in ecological and evolutionary studies

2015

View full text Add to dashboard Cite

Summary1. Body condition indices, measures of body 'plumpness' or mass relative to frame size, are often used as a proxy for lipid reserves or fitness-related traits of animals and assumed to be positively related to fitness. 2. The quantification and analysis of body condition indices has been the subject of debate for decades. Here, we summarize three additional concerns with the use of body condition indices. 3. First, body condition index is often poorly correlated with lipid content in animals. Second, even if body condition index and lipid content are correlated, lipid content of an animal may not be the most important aspect of body composition influencing fitness. Finally, neither body condition index nor lipid reserves are likely to be directly positively related to fitness in animals, as many animals homeostatically regulate intermediate levels of condition index or lipid reserves, with both higher and lower values incurring fitness costs. 4. A wide range of analytical methods, including some relatively inexpensive and simple measures, are available for more detailed measures of animal body composition or fitness-related traits. Replacing body condition indices with more direct measures of body composition -even relatively simple measures -can inform understanding of the physiological mechanisms underlying animal responses in a wide range of behavioural, ecological and evolutionary studies.

show abstract

Development of a Univariate Membrane-Based Mid-Infrared Method for Protein Quantitation and Total Lipid Content Analysis of Biological Samples

Cited by 28 publications

References 45 publications

In vitro characterization of a novel Isu homologue from Drosophila melanogaster for de novo FeS-cluster formation

In vitro characterization of a novel Isu homologue from Drosophila melanogaster for de novo FeS-cluster formation

Quantification of hydrolyzed peptides and proteins by amino acid fluorescence

Moving beyond body condition indices as an estimate of fitness in ecological and evolutionary studies

Contact Info

Product

Resources

About