TY - JOUR
T1 - Development of a Robust Ultrasonic-Based Sample Treatment to Unravel the Proteome of OCT-Embedded Solid Tumor Biopsies
AU - Jorge, Susana
AU - Capelo, José L.
AU - Laframboise, William
AU - Dhir, Rajiv
AU - Lodeiro, Carlos
AU - Santos, Hugo M.
N1 - The PROTEOMASS Scientific Society is acknowledged for the funding provided to the Laboratory for Biological Mass Spectrometry Isabel Moura. We acknowledge the funding provided by the Associate Laboratory for Green Chemistry LAQV, which is financed by national funds from FCT/MEC (UID/QUI/50006/2019).
H.M.S. is funded by the FCT 2015 Investigator Program (IF/00007/2015).
S.J. thanks FCT/MEC (Portugal) for her research contract as a Ph.D. student with the grant SFRH/BD/120537/2016.
This project utilized the University of Pittsburgh Hillman Cancer Center shared resource facility (Cancer Genomics Facility), supported in part by award P30CA047904 (W.L.).
PY - 2019/1/1
Y1 - 2019/1/1
N2 - An effective three-step proteomics workflow is proposed to overcome the pitfalls caused by polymers present in optimum cutting temperature (OCT)-embedded tissue during its preparation for mass spectrometry analysis. First, the OCT-embedded tissue biopsies are cleaned using ethanol and water in a sequential series of ultrasonic washes in an ultrasound bath (35 kHz ultrasonic frequency, 100% ultrasonic amplitude, 2 min of ultrasonic duty time). Second, a fast ultrasonic-assisted extraction of proteins is done using an ultrasonic probe (30 kHz ultrasonic frequency, 50% ultrasonic amplitude, 2 min of ultrasonic duty time, 1 mm diameter tip). Third, a rapid ultrasonic digestion of complex proteomes is performed using a microplate horn assembly device (20 kHz ultrasonic frequency, 25% ultrasonic amplitude, 4 min of ultrasonic duty time). As a proof of concept, the new workflow was applied to human normal and tumor kidney biopsies including chromophobe renal cell carcinomas (chRCCs) and renal oncocytomas (ROs). A successful cluster of proteomics profiles was obtained comprising 511 and 172 unique proteins found in chRCC and RO samples, respectively. The new method provides high sample throughput and comprehensive protein recovery from OCT samples.
AB - An effective three-step proteomics workflow is proposed to overcome the pitfalls caused by polymers present in optimum cutting temperature (OCT)-embedded tissue during its preparation for mass spectrometry analysis. First, the OCT-embedded tissue biopsies are cleaned using ethanol and water in a sequential series of ultrasonic washes in an ultrasound bath (35 kHz ultrasonic frequency, 100% ultrasonic amplitude, 2 min of ultrasonic duty time). Second, a fast ultrasonic-assisted extraction of proteins is done using an ultrasonic probe (30 kHz ultrasonic frequency, 50% ultrasonic amplitude, 2 min of ultrasonic duty time, 1 mm diameter tip). Third, a rapid ultrasonic digestion of complex proteomes is performed using a microplate horn assembly device (20 kHz ultrasonic frequency, 25% ultrasonic amplitude, 4 min of ultrasonic duty time). As a proof of concept, the new workflow was applied to human normal and tumor kidney biopsies including chromophobe renal cell carcinomas (chRCCs) and renal oncocytomas (ROs). A successful cluster of proteomics profiles was obtained comprising 511 and 172 unique proteins found in chRCC and RO samples, respectively. The new method provides high sample throughput and comprehensive protein recovery from OCT samples.
KW - chromophobe renal cell carcinoma
KW - label-free quantification
KW - mass spectrometry
KW - OCT-embedded tissues
KW - renal oncocytoma
KW - ultrasound energy
UR - http://www.scopus.com/inward/record.url?scp=85068054527&partnerID=8YFLogxK
U2 - 10.1021/acs.jproteome.9b00248
DO - 10.1021/acs.jproteome.9b00248
M3 - Article
C2 - 31173681
AN - SCOPUS:85068054527
SN - 1535-3893
VL - 18
SP - 2979
EP - 2986
JO - Journal Of Proteome Research
JF - Journal Of Proteome Research
IS - 7
ER -