Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA043439

Local Sample ID:	philic_16mar16-020-r001
Subject ID:	SU000814
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Species Group:	Human

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Subject:

Subject ID:	SU000814
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Species Group:	Human

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
philic_16mar16-020-r001	SA043439	FL008953	Quick Progress	groups
philic_16mar16-020-r001	SA043439	FL008953	1	rep

Collection:

Collection ID:	CO000808
Collection Summary:	In order to analyze the metabolites of clonal PCs (intracellular) and BM plasma (extracellular) separately, they have to be separated out from the BM samples. Thus, upon acquiring the BM samples from patients they will be placed in centrifuged at 2500 rpm for 10 minutes to separate out the plasma from the cellular fraction. The separated BM plasma is then stored in separate vials and snap frozen under liquid nitrogen for 20 seconds before storing at -80οC for further analysis. The leftover cellular component present as a pellet will be washed and reconstituted with an equal volume of RPMI 1640 medium. Erythrocytes are lysed using ammonium chloride lysing solution. After incubation on ice for 5 min, the cell suspension is diluted with RPMI medium. The cells are again pelleted by centrifugation and then suspended in RoboSep buffer (250ml PBS, 2% BSA, 1mM EDTA). The clonal CD138 positive PCs are purified using positive selection by mixing the cells with a CD138 positive selection cocktail and anti-CD138 magnetic-activated cell separation microbeads (ROBOSEP™ cell separation system, StemCell Technologies Inc) in the automated RoboSep cell separation system. The purified samples containing only CD138 positive cells are re‐suspended and then centrifuged to form a cell pellet. The goal will be to obtain at least 1-2 x 107 clonal PCs per sample. The cell pellet will be snap frozen under liquid nitrogen for 20 seconds before storing at -80οC for further analysis. Both the BM plasma samples as well as the clonal PCs pellet will be provided to the metabolomics core for sample preparation for LC-MS analysis. For Aim 2, we will be using stored BM samples from SMM patients that have already had their BM plasma and clonal PCs separated from each other and stored at -80οC. It is important to note that all these samples were collected and frozen in a timely manner. Furthermore consistency in sample collection, storage and processing is imperative for the optimal conduct of metabolomics-based experiments. This ensures that all samples being analyzed and compared with each other would have been manipulated similarly, limiting any handling biases. All patient samples in the Predolin Foundation Biobank were collected and stored by following a standardized operating procedure. BM samples were processed for BM plasma separation and clonal PCs enrichment but were then immediately snap frozen for storage at -80οC within approximately 3 hours of collection from the patient. All samples were collected in patients who have been fasting for at least 6 to 8 hours prior. To further ensure consistency in our analysis of this study, we will only use samples that have never been thawed since initial storage to preserve the stability of the metabolites originally present in the samples.
Sample Type:	Bone marrow

Treatment:

Treatment ID:	TR000828
Treatment Summary:	We will use matched BM plasma and purified clonal marrow PCs from the BM samples of SMM patients collected at the time of their diagnosis and stored within the Mayo Clinic Predolin Foundation Biobank. We will select BM samples from patients with SMM who progressed to MM within 2 years of their samples being collected and stored; this will constitute the high risk SMM group. We will also select BM samples from SMM patients who have not progressed to MM within at least 2 years of follow up of their samples being collected and stored; this will constitute the standard risk SMM group. The strength of this approach is that we will utilize stored SMM samples from one of the most comprehensively characterized monoclonal gammopathy biobanks available. Secondly, all samples will be obtained from collection dates at least 2 years prior in order to ensure adequate follow-up time to assess their current clinical status. This would avoid the need to prospectively collect samples from SMM patients and clinically follow them for a number of years before we are able to gauge who were clinically high or standard risk for progression. There are over 700 SMM patients who have had their BM samples collected and stored in the biobank from 1996 to 2013; more than half these patients have progressed to MM.

Treatment ID:

TR000828

Treatment Summary:

We will use matched BM plasma and purified clonal marrow PCs from the BM samples of SMM patients collected at the time of their diagnosis and stored within the Mayo Clinic Predolin Foundation Biobank. We will select BM samples from patients with SMM who progressed to MM within 2 years of their samples being collected and stored; this will constitute the high risk SMM group. We will also select BM samples from SMM patients who have not progressed to MM within at least 2 years of follow up of their samples being collected and stored; this will constitute the standard risk SMM group. The strength of this approach is that we will utilize stored SMM samples from one of the most comprehensively characterized monoclonal gammopathy biobanks available. Secondly, all samples will be obtained from collection dates at least 2 years prior in order to ensure adequate follow-up time to assess their current clinical status. This would avoid the need to prospectively collect samples from SMM patients and clinically follow them for a number of years before we are able to gauge who were clinically high or standard risk for progression. There are over 700 SMM patients who have had their BM samples collected and stored in the biobank from 1996 to 2013; more than half these patients have progressed to MM.

Sample Preparation:

Sampleprep ID:	SP000821
Sampleprep Summary:	The metabolite profiling as well as sample preparations will be undertaken at the Mayo Clinic Metabolomics Resource Core. The matched BM plasma and clonal PCs pellets from the stored patient samples will undergo metabolite extraction and analysis by LC-MS. They will be deproteinized with cold methanol (1:4 ratio) and centrifuged at 15,000 g for 20 minutes at 4οC. The supernatants will be divided into 2 aliquots and dried down under a stream of nitrogen for 90 minutes and stored at -20C until ready for analysis. These samples will undergo preparation for metabolite separation and analysis on a Time-of-Flight (TOF) Mass Spectrometer (Agilent Technologies 6220 TOF) coupled with an Ultra High Pressure Liquid Chromatograph (Waters Acquity). The dried metabolites will be re‐suspended in H2O/acetonitrile solution containing injection standards. Profiling data will be acquired under both positive and negative electrospray ionization modes separately over a scan range of 50 - 1200 m/z at a resolution of 10,000. Metabolite separation will be achieved using two columns of differing polarity, a hydrophilic interaction column (HILIC, ethylene-bridged hybrid 2.1 x 150 mm, 1.7 mm; Waters) and a reversed-phase C18 column (high-strength silica 2.1 x 150 mm, 1.8 mm; Waters). For positive ion detection, mobile phase A will contain 100% water with 0.1% formic acid and mobile phase B will contain 100% methanol with 0.1% formic acid. For negative ion detection, formic acid will be replaced with 0.1% ammonium bicarbonate. For each column, the run time will be 20 min using a flow rate of 400 microL/min. A total of four runs per sample will be performed to give maximum coverage of metabolites. Samples will be injected in triplicate with blank injections between samples. Quality control samples, made up of a subset of samples from the study will be injected several times during a run.

Sampleprep ID:

SP000821

Sampleprep Summary:

The metabolite profiling as well as sample preparations will be undertaken at the Mayo Clinic Metabolomics Resource Core. The matched BM plasma and clonal PCs pellets from the stored patient samples will undergo metabolite extraction and analysis by LC-MS. They will be deproteinized with cold methanol (1:4 ratio) and centrifuged at 15,000 g for 20 minutes at 4οC. The supernatants will be divided into 2 aliquots and dried down under a stream of nitrogen for 90 minutes and stored at -20C until ready for analysis. These samples will undergo preparation for metabolite separation and analysis on a Time-of-Flight (TOF) Mass Spectrometer (Agilent Technologies 6220 TOF) coupled with an Ultra High Pressure Liquid Chromatograph (Waters Acquity). The dried metabolites will be re‐suspended in H2O/acetonitrile solution containing injection standards. Profiling data will be acquired under both positive and negative electrospray ionization modes separately over a scan range of 50 - 1200 m/z at a resolution of 10,000. Metabolite separation will be achieved using two columns of differing polarity, a hydrophilic interaction column (HILIC, ethylene-bridged hybrid 2.1 x 150 mm, 1.7 mm; Waters) and a reversed-phase C18 column (high-strength silica 2.1 x 150 mm, 1.8 mm; Waters). For positive ion detection, mobile phase A will contain 100% water with 0.1% formic acid and mobile phase B will contain 100% methanol with 0.1% formic acid. For negative ion detection, formic acid will be replaced with 0.1% ammonium bicarbonate. For each column, the run time will be 20 min using a flow rate of 400 microL/min. A total of four runs per sample will be performed to give maximum coverage of metabolites. Samples will be injected in triplicate with blank injections between samples. Quality control samples, made up of a subset of samples from the study will be injected several times during a run.

Combined analysis:

Analysis ID	AN001254	AN001255	AN001256	AN001257
Analysis type	MS	MS	MS	MS
Chromatography type	Reversed phase	Reversed phase	HILIC	HILIC
Chromatography system	Agilent 1290 Infinity	Agilent 1290 Infinity	Agilent 1290 Infinity	Agilent 1290 Infinity
Column	Waters Acquity HSS C18 (150 x 2.1mm,1.8um)	Waters Acquity HSS C18 (150 x 2.1mm,1.8um)	Waters Acquity BEH Amide (150 x 2.1mm,1.7um)	Waters Acquity BEH Amide (150 x 2.1mm,1.7um)
MS Type	ESI	ESI	ESI	ESI
MS instrument type	QTOF	QTOF	QTOF	QTOF
MS instrument name	Agilent 6550 QTOF	Agilent 6550 QTOF	Agilent 6550 QTOF	Agilent 6550 QTOF
Ion Mode	POSITIVE	NEGATIVE	POSITIVE	NEGATIVE
Units	intensity	intensity	intensity	intensity

Chromatography:

Chromatography ID:	CH000874
Instrument Name:	Agilent 1290 Infinity
Column Name:	Waters Acquity HSS C18 (150 x 2.1mm,1.8um)
Chromatography Type:	Reversed phase

Chromatography ID:	CH000875
Instrument Name:	Agilent 1290 Infinity
Column Name:	Waters Acquity BEH Amide (150 x 2.1mm,1.7um)
Chromatography Type:	HILIC

MS:

MS ID:	MS001147
Analysis ID:	AN001254
Instrument Name:	Agilent 6550 QTOF
Instrument Type:	QTOF
MS Type:	ESI
Ion Mode:	POSITIVE

MS ID:	MS001148
Analysis ID:	AN001255
Instrument Name:	Agilent 6550 QTOF
Instrument Type:	QTOF
MS Type:	ESI
Ion Mode:	NEGATIVE

MS ID:	MS001149
Analysis ID:	AN001256
Instrument Name:	Agilent 6550 QTOF
Instrument Type:	QTOF
MS Type:	ESI
Ion Mode:	POSITIVE

MS ID:	MS001150
Analysis ID:	AN001257
Instrument Name:	Agilent 6550 QTOF
Instrument Type:	QTOF
MS Type:	ESI
Ion Mode:	NEGATIVE