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Preserving the phosphoproteome of clinical biopsies using a quick freeze collection device

ABSTRACT: The current gold standard for collecting and processing cancer biopsies is to flash freeze tumor samples in liquid nitrogen. However, in many clinical settings liquid nitrogen is not readily available, and neither the personnel nor the infrastructure is generally available to rapidly process the tumor samples. As a result, tumors are often subjected to prolonged ischemia and/or chemical fixatives, altering the phosphoproteome such that it may no longer reflect the true in vivo state of the tumor. There is a need for an economical, single-use device that can be stored at room temperature then activated at point of care to rapidly freeze the specimen. Our proof-of-concept quick-freeze prototype device focused on key requirements including cooling performance, device safety, minimizing use error, and the ability to ship via existing cold chain logistics. We have demonstrated that our device can cool a core sample below 0°C in less than 70 seconds, below -8°C in less than 150 seconds, and maintain that sample below 0°C for greater than 70 minutes. To demonstrate feasibility, the performance of our prototype was benchmarked against flash freezing in liquid nitrogen using melanoma-bearing PDX mice as a model system. After subjecting the mice to total body irradiation to elicit a phosphosignaling response in the DNA damage response pathway, tumors were harvested and quadrisected with two parts of the tumor snap frozen in liquid nitrogen, and the remaining two parts rapidly cooled in the prototype quick-freeze biospecimen containers for 1 hour. Phosphoproteins were profiled by LC-MS/MS. The prototype freeze device showed feasibility with bias within acceptable limits and slightly higher variability for a subset of phosphopeptides compared to flash freezing. The prototype device forms the framework for development of a commercial device that will improve tissue biopsy preservation for measurement of important phospho-signaling molecules.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Melanocyte

DISEASE(S): Mucosal Melanoma

SUBMITTER: Jacob Kennedy

LAB HEAD: Amanda G Paulovich

PROVIDER: PXD029844 | Pride | 2022-07-18

REPOSITORIES: Pride

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			Action	DRS
	Freeze_PDX_TMT_IMAC_bRP_LCMSMS_MaxQuant_txt.zip	Other
	JK040820_041320_PCS_Freeze_bRP_IMAC_TMT_Gr01_rep2_F01.raw	Raw
	JK040820_041320_PCS_Freeze_bRP_IMAC_TMT_Gr01_rep2_F02.raw	Raw
	JK040820_041320_PCS_Freeze_bRP_IMAC_TMT_Gr01_rep2_F03.raw	Raw
	JK040820_041320_PCS_Freeze_bRP_IMAC_TMT_Gr01_rep2_F04.raw	Raw

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Project description:Four protocols were adapted and compared to enrich extracellular matrix proteins from mouse skin: 1. compartmental enrichment; 2. chemical decellularization; 3. enzymatic decellularization using phospholipase A2; 4. quantitative detergent solubility profiling (QDSP). All four protocols were started the same day, each using 35 mg of frozen back skin taken from the same initial back skin piece. The experiment was repeated three times starting from three different mice. Compartmental enrichment: skin samples were processed using the CNMCS commercial kit. Briefly, the skin tissue was homogenized in buffer C and successively incubated under agitation in buffers W, N, M and CS supplemented with the protease inhibitors from the kit. Extraction buffer C and insoluble pellet were kept aside, flash-frozen in liquid nitrogen immediately. Chemical decellularization: skin samples were cut into small pieces and successively incubated in buffers containing NaCl, SDS/sodium deoxycholate, Triton X-100 and GuHCl. Extractions buffers as well as the final insoluble pellet were kept aside, flash-frozen in liquid nitrogen immediately. Enzymatic decellularization: skin samples were cut into small pieces and successively incubated in buffers containing NaCl, phosopholipase A2/sodium deoxycholate. Extraction buffers as well as the insoluble pellet were kept aside, flash-frozen in liquid nitrogen immediately. QDSP: skin samples were homogenized in sterile PBS and successively incubated in 3 buffers containing increasing concentrations in detergents (including SDS, sodium deoxycholate, NP-40) as previously described. Extraction buffers as well as the insoluble pellet were kept aside, flash-frozen in liquid nitrogen immediately. For comparison with the four protocols described above, total skin lysates were also prepared for two of the mice. In this case, 20 mg of mouse skin were homogenized and incubated in a lysis buffer containing SDS, β-mercaptoethanol. Samples were centrifuged and the supernatant was kept aside, flash-frozen in liquid nitrogen immediately.

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Preserving the phosphoproteome of clinical biopsies using a quick freeze collection device

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