General recommendations for metabolomics analysis:

Sample collection, storage, labeling and shipping guideline

A discussion is strongly recommended before a pilot study.

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(1) Sample Collection

Samples must be grown, harvested, and processed under strictly identical conditions. To prevent degradation, maintain samples at the lowest possible temperature throughout all processing steps. For reliable results, consistent sample collection is essential. Minimize process variables during sampling and maintain strict uniformity in collection site, time, and handling (e.g., collection method, procedure, sampling time, and time spent above -80°C).

Minimize processing time during collection, preparation, separation, and cryopreservation to reduce potential degradation. Because metabolic activity continues in biological samples with active enzymes, metabolite levels can change rapidly and significantly. Therefore, to obtain a representative metabolic profile for accurate and meaningful group comparisons, metabolism must be quenched immediately and effectively. Finally, avoid all additives, detergents, and preservatives, as some are incompatible with mass spectrometry and may interfere with downstream analyses. Use high-quality polypropylene cryovials or Eppendorf tubes (2.0 or 1.5 mL) with external screw caps for sample aliquoting and storage. Biohazardous and radioactive samples must not be processed.

(2) Sample Storage

Following sample collection, rapid freezing in liquid nitrogen is recommended for all sample types prior to long-term storage at -80℃. If liquid nitrogen is unavailable, samples should be placed at -80℃ immediately after collection. Liquid samples must be stored upright before freezing to ensure they remain at the bottom of the tube. To minimize freeze-thaw cycles for metabolomic samples, aliquot samples prior to storage if multiple analyses are anticipated.

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(3) Sample labeling

All sample tubes must be clearly and uniquely labeled. Label the tubes with a permanent marker or cryogenic labels before freezing, while they are still at room temperature. It is extremely difficult to write on or label frozen tubes. Non-cryogenic labels are prone to detaching during storage at -80℃; use them with extreme caution. To protect labels from organic solvents and extreme cold, apply clear tape over them. Ensure all labels are legible and securely affixed.

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(4) Sample shipping

Prior to shipping, please ensure the package contains enough dry ice to prevent sample thawing or degradation during transport. Include a hard copy of the complete sample list, containing all available sample information, with the shipment. Arrange the samples in the freezer box according to the order specified on the sample list.

Ship samples to:

Dr. Jung-Lee Lin

Mass Core Facility, Genomic Research Center, Academia Sinica

Room 2L13

128 Academia Road, Section 2, Nankang,

Taipei, 115, Taiwan

E-mail: harrylin @gate.sinica.edu.tw

TEL: (02)2789-8754

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(5) Sample requirement

Serum/Plasma 300μL

Cultured Cell 1‐10 million cells

Tissue 10 to 50mg

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(6) Normalization

Users are responsible for measuring the total protein content of each sample. It is crucial that all samples are measured on the same plate. These values will be used to normalize the data after sample analysis.