Summary of Study ST000543

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR000397. The data can be accessed directly via it's Project DOI: 10.21228/M8R013 This work is supported by NIH grant, U2C- DK119886.

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Study ID	ST000543
Study Title	High Resolution orbittrap Mass Spectrometer to measure low abundance isotope enrichment in individual muscle proteins
Study Type	isotope encrichment and comparison of mass spectrometer platforms
Study Summary	Stable isotope-labeled amino acids have long been used to measure the fractional synthesis rate of proteins, although the mass spectrometry platforms used for such analyses have changed throughout the years. More recently, tandem mass spectrometers such as triple quadrupoles have been accepted as the standard platform for enrichment measurement due to their sensitivity and the enhanced specificity offered by multiple reaction monitoring (MRM) experiments. The limit in the utility of such platforms for enrichment analysis occurs when measuring very low levels of enrichment from small amounts of sample, particularly proteins isolated from two-dimensional gel electrophoresis (2D-GE), where interference from contaminant ions impact the sensitivity of the measurement. We therefore applied a high resolution orbitrap mass spectrometer to the analysis of [ring-13C6]-phenylalanine enrichment in individual muscle proteins isolated with 2D-GE. Comparison of samples analyzed on both platforms revealed that the high resolution MS has significantly improved sensitivity relative to the triple quadrupole MS at very low-level enrichments due to its ability to resolve interferences in the m/z dimension.
Institute	Mayo Clinic
Department	Endocrinology
Laboratory	Mayo Clinic Metabolomics Resource Core
Last Name	Nair
First Name	Sreekumaran
Address	200 First Street SW, Rochester, MN 55905
Email	Nair.K@mayo.edu
Phone	507-285-2415
Submit Date	2016-07-22
Analysis Type Detail	LC-MS
Release Date	2017-07-10
Release Version	1

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Project:

Project ID:	PR000397
Project DOI:	doi: 10.21228/M8R013
Project Title:	Application of high-resolution mass spectrometry to measure low abundance isotope enrichment in individual muscle proteins
Project Type:	MS targeted analysis of [ring-13C6]-phenylalanine
Project Summary:	Comparison of mass spectrometer methods to the analysis of [ring-13C6]-phenylalanine enrichment in individual muscle proteins isolated with 2D-GE
Institute:	Mayo Clinic
Department:	Endocrinology
Laboratory:	Mayo Clinic Metabolomics Resource Core
Last Name:	Nair
First Name:	Sreekumaran
Address:	200 First Street SW, Rochester, MN 55905
Email:	Nair.K@mayo.edu
Phone:	507-285-2415

Subject:

Subject ID:	SU000565
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Species Group:	Human

Factors:

Subject type: Human; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id	local_sample_id	group	time (mins)
SA028063	R13A-180	A	180
SA028064	R5A-180	A	180
SA028065	R3A-180	A	180
SA028066	R17A-180	A	180
SA028067	R2A-180	A	180
SA028068	R15A-180	A	180
SA028069	R10A-180	A	180
SA028070	R12A-180	A	180
SA028071	R6A-180	A	180
SA028072	R16A-180	A	180
SA028073	R3A-480	A	480
SA028074	R17A-480	A	480
SA028075	R16A-480	A	480
SA028076	R13A-480	A	480
SA028077	R10A-480	A	480
SA028078	R6A-480	A	480
SA028079	R5A-480	A	480
SA028080	R15A-480	A	480
SA028081	R2A-480	A	480
SA028082	R12A-480	A	480
SA028083	R5B-180	B	180
SA028084	R2B-180	B	180
SA028085	R6B-180	B	180
SA028086	R3B-180	B	180
SA028087	R10B-180	B	180
SA028088	R16B-180	B	180
SA028089	R12B-180	B	180
SA028090	R13B-180	B	180
SA028091	R15B-180	B	180
SA028092	R17B-180	B	180
SA028093	R16B-480	B	480
SA028094	R5B-480	B	480
SA028095	R15B-480	B	480
SA028096	R10B-480	B	480
SA028097	R3B-480	B	480
SA028098	R2B-480	B	480
SA028099	R17B-480	B	480
SA028100	R13B-480	B	480
SA028101	R12B-480	B	480
SA028102	R6B-480	B	480
SA028103	R3C-180	C	180
SA028104	R2C-180	C	180
SA028105	R3C-480	C	480
SA028106	R2C-480	C	480

Showing results 1 to 44 of 44

Showing results 1 to 44 of 44

Collection:

Collection ID:	CO000559
Collection Summary:	Percutaneous needle biopsies of the vastus lateralis muscle were performed under local anesthesia at 180 min and 480 min into the infusion [3;21]. The studies were repeated 65-80 days following the first study. Explanation of study design factors: Study A is the first study period, Study B is the second study conducted after 655-80 days after the first, Time 180 is the first muscle biopsy within the given study period, Time 480 is the second muscle biopsy within the given study period. Study C is the third study conducted after the second for a few subjects.
Sample Type:	Muscle

Treatment:

Treatment ID:	TR000579
Treatment Summary:	We performed studies in healthy study participants in the Mayo Clinic Clinical Research Unit (CRU) after obtaining informed consent as a protocol approved by the Institutional Review Board. In ten healthy human study participants (age = 57.1 ± 20.24 yrs; M:F ratio = 1.5; and BMI = 26.80 ± 3.38 kg/mL), after overnight fast, we infused [ring-13C6]-phenylalanine at a rate of 1 mg/kg FFM/hr for eight hours following a priming dose to achieve an early isotope plateau [19;20].

Treatment ID:

TR000579

Treatment Summary:

We performed studies in healthy study participants in the Mayo Clinic Clinical Research Unit (CRU) after obtaining informed consent as a protocol approved by the Institutional Review Board. In ten healthy human study participants (age = 57.1 ± 20.24 yrs; M:F ratio = 1.5; and BMI = 26.80 ± 3.38 kg/mL), after overnight fast, we infused [ring-13C6]-phenylalanine at a rate of 1 mg/kg FFM/hr for eight hours following a priming dose to achieve an early isotope plateau [19;20].

Sample Preparation:

Sampleprep ID:	SP000572
Sampleprep Summary:	From the human muscle biopsies, 20-30 mg of quadriceps muscle was homogenized in urea buffer (9.8M urea, 4% CHAPS) and skeletal muscle mitochondria separated using a differential centrifugation [22]. Individual proteins were isolated from the mixture by performing large, high-resolution, 2D-GE [23]. Approximately 200 μg of each protein sample were dissolved in lysis buffer to a final volume of 450 μl. These samples were used to rehydrate 24-cm, pH 4–7 and 6–9, immobilized pH gradient (IPG) strips (Bio-Rad Laboratories, Hercules, CA) in a rehydration tray overnight. The rehydrated IPG strips were subjected to isoelectric focusing in a Protean IEF Cell (Bio-Rad) using a three-step protocol: i) the focusing was achieved with an initial step of 250 V for 15 min; ii) continued with a maximum of 10,000 V increased linearly from 250 V over 6 h; and iii) continued at 10,000 V for 6 h. The cell temperature was kept at 20°C with a maximum current of 50 μA per strip. The IPG strips were then equilibrated for the SDS-PAGE in a two-step equilibration using 5 mL of equilibration buffer per strip (6 M urea, 2% SDS, 0.375 M Tris·HCl, pH 8.8, and 20% glycerol) with 130 mM DTT in the first step and 135 mM iodoacetamide in the second step. The equilibration steps were done in an equilibration tray for 10 min each on a rotary shaker at room temperature. The second-dimension separation by subunit molecular weight was performed by vertical 12%, 24 × 20-cm dimension SDS-PAGE (Ettan DALT system; GE Healthcare Bio-Sciences, Piscataway, NJ). The IPG strips were mounted into the IPG well with molten agarose and then run at 75 V for 24 h or until the dye front reached the bottom of the gel. The protein gel spots were visualized by staining with Coomassie blue (GelCode Blue Stain Reagent; Pierce, Rockford, IL). Spots were excised from the gel, placed in glass vials, and washed several times with water. An additional 3 mL of HPLC water (Fisher Scientific) was added to each vial, and the gel spot samples were placed on a rocking shaker for 60 min. The proteins were then hydrolyzed at 120 °C for 18 h with 6 M HCl. The following day, the gel spot samples were centrifuged for 5 min at 3,000 rpm and 4 °C, after which 2 mL of water was added to each vial and vortexed. To prepare the AG-50x8 cation exchange column, the resin was rinsed with 4 mL of 4M ammonium hydroxide (NH4OH), followed by 4 rinses with 5 mL water. The column resin was regenerated with 4 mL of 4M HCl and rinsed with 5 mL of 0.1M HCl. The gel spot samples (approx. 2 mL) were transferred to the prepared AG-50 columns, the column was rinsed 4X with 4 mL of HPLC water, and amino acids were eluted into washed glass vials with three 1 mL washes of 4M NH4OH. The eluents were dried overnight in a speed-vac without heat. Amino acids were derivatized with 50 µL of 4M HCl in dry isobutanol at 85 °C for 45 min and dried under nitrogen. For LC-MS/MS analyses, 40 µL of 5% acetonitrile in water was added to each vial, vortexed and transferred to autosampler vials.

Sampleprep ID:

SP000572

Sampleprep Summary:

From the human muscle biopsies, 20-30 mg of quadriceps muscle was homogenized in urea buffer (9.8M urea, 4% CHAPS) and skeletal muscle mitochondria separated using a differential centrifugation [22]. Individual proteins were isolated from the mixture by performing large, high-resolution, 2D-GE [23]. Approximately 200 μg of each protein sample were dissolved in lysis buffer to a final volume of 450 μl. These samples were used to rehydrate 24-cm, pH 4–7 and 6–9, immobilized pH gradient (IPG) strips (Bio-Rad Laboratories, Hercules, CA) in a rehydration tray overnight. The rehydrated IPG strips were subjected to isoelectric focusing in a Protean IEF Cell (Bio-Rad) using a three-step protocol: i) the focusing was achieved with an initial step of 250 V for 15 min; ii) continued with a maximum of 10,000 V increased linearly from 250 V over 6 h; and iii) continued at 10,000 V for 6 h. The cell temperature was kept at 20°C with a maximum current of 50 μA per strip. The IPG strips were then equilibrated for the SDS-PAGE in a two-step equilibration using 5 mL of equilibration buffer per strip (6 M urea, 2% SDS, 0.375 M Tris·HCl, pH 8.8, and 20% glycerol) with 130 mM DTT in the first step and 135 mM iodoacetamide in the second step. The equilibration steps were done in an equilibration tray for 10 min each on a rotary shaker at room temperature. The second-dimension separation by subunit molecular weight was performed by vertical 12%, 24 × 20-cm dimension SDS-PAGE (Ettan DALT system; GE Healthcare Bio-Sciences, Piscataway, NJ). The IPG strips were mounted into the IPG well with molten agarose and then run at 75 V for 24 h or until the dye front reached the bottom of the gel. The protein gel spots were visualized by staining with Coomassie blue (GelCode Blue Stain Reagent; Pierce, Rockford, IL). Spots were excised from the gel, placed in glass vials, and washed several times with water. An additional 3 mL of HPLC water (Fisher Scientific) was added to each vial, and the gel spot samples were placed on a rocking shaker for 60 min. The proteins were then hydrolyzed at 120 °C for 18 h with 6 M HCl. The following day, the gel spot samples were centrifuged for 5 min at 3,000 rpm and 4 °C, after which 2 mL of water was added to each vial and vortexed. To prepare the AG-50x8 cation exchange column, the resin was rinsed with 4 mL of 4M ammonium hydroxide (NH4OH), followed by 4 rinses with 5 mL water. The column resin was regenerated with 4 mL of 4M HCl and rinsed with 5 mL of 0.1M HCl. The gel spot samples (approx. 2 mL) were transferred to the prepared AG-50 columns, the column was rinsed 4X with 4 mL of HPLC water, and amino acids were eluted into washed glass vials with three 1 mL washes of 4M NH4OH. The eluents were dried overnight in a speed-vac without heat. Amino acids were derivatized with 50 µL of 4M HCl in dry isobutanol at 85 °C for 45 min and dried under nitrogen. For LC-MS/MS analyses, 40 µL of 5% acetonitrile in water was added to each vial, vortexed and transferred to autosampler vials.

Combined analysis:

Analysis ID	AN000825
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Thermo Dionex Ultimate 3000
Column	Zorbax Extended C18
MS Type	ESI
MS instrument type	Orbitrap
MS instrument name	Thermo Q Exactive Plus Orbitrap
Ion Mode	POSITIVE
Units	mole percent enrichment

Chromatography:

Chromatography ID:	CH000589
Chromatography Summary:	UPLC separations were performed with a Dionex UltiMate 3000 system (ThermoFisher Scientific) and a Zorbax Extended C18 column (Agilent; 5 cm × 2.1 mm, 1.8 µm). Solvent A was 99% water (Fisher Scientific), 1% acetonitrile (Fisher Scientific) with 0.1% formic acid (SigmaAldrich), and solvent B was 99% acetonitrile, 1% water and 0.1% formic acid. The gradient was as follows: 0-6 min: 5-20% B; 6-10 min: 20-95% B; 10-12 min: 95% B; 12-13 min: 95-5% B, at a flow rate of 0.3 mL min−1. An injection volume of 5 µL was used. The UPLC was connected to the ion source through the diverter valve. The HESI ion source was operated with +3.5 kV spray voltage and probe heater temperature of 300 °C. Sheath, auxiliary and spare gas were 47.5, 11.25 and 1.0 (arb. units), respectively. The capillary temperature was maintained at 275 °C.
Instrument Name:	Thermo Dionex Ultimate 3000
Column Name:	Zorbax Extended C18
Flow Gradient:	0-6 min: 5-20% B; 6-10 min: 20-95% B; 10-12 min: 95% B; 12-13 min: 95-5% B
Flow Rate:	0.3ml/min
Solvent A:	99% water/1% acetonitrile; 0.1% formic acid
Solvent B:	99% acetonitrile/1% water; 0.1% formic acid
Chromatography Type:	Reversed phase

MS:

MS ID:	MS000726
Analysis ID:	AN000825
Instrument Name:	Thermo Q Exactive Plus Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
Ion Mode:	POSITIVE