Using MALDI-TOF MS Coupled with a High-Mass Detector to Directly Analyze Intact Proteins in Thyroid Tissues

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May 11, 2018

Authors

Shan-Shan Wang1, Yun-Jun Wang2,3, Jing Zhang1, Jun Xiang2,3, Tuan-Qi Sun2,3,& Yin-Long Guo1

Organizations

State Key Laboratory of Organometallic Chemistry and National Center for Organic Mass Spectrometry in Shanghai, Center for Excellence in Molecular Synthesis, Shanghai Institute of Organic Chemistry, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai 200032, China;
Department of Head and Neck Surgery, Fudan University Shanghai Cancer Center, Shanghai 200032, China;
Department of Oncology, Shanghai Medical College, Fudan University, Shanghai 200032, China

Abstract

Protein analysis is vital for biological and clinical research, but the measurement of unseparated, intact and high-mass proteins is also a challenging task by mass spectrometry-based methods. Here, we present a protocol for rapid and high-throughput analysis of intact proteins in tissue samples using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) combined with a high-mass detector platform. The method involves tissue specimens that undergo a simple protein extraction before MALDI-MS analysis. Using this method, the high abundance proteins in human thyroid carcinoma and paracarcinoma tissues were successfully investigated, and the mass spectra of the tissues of the 30 illustrated thyroid cancers showed remarkable differences. The peak intensity revealed a significant increase in human albumin in thyroid carcinoma tissues (p<0.05). To validate the feasibility and credibility of this method, label-free proteomics quantitative analysis and Western blotting were used to relatively quantify the proteins in these tissues. Those results demonstrated a nearly 3-fold difference in human albumin levels between thyroid carcinoma and para-carcinoma tissues, which were consistent with the results of our method. The advantages of our method are easy sample handling, remarkable reproducibility and the ability to analyze high-mass proteins without digestion, which make them have the potential to be used in biological research and in clinical practice.

CovalX Technology Used (Click each option to learn more)

HM3

Outcomes

Thyroid tissue specimens were subjected to simple protein extraction before being subjected to MALDI-MS analysis. This method of analysis allowed researchers to investigate the large number of proteins present in human thyroid carcinoma and para-carcinoma (normal) tissues. The use of MALDI-MS allowed for the analysis of high-mass proteins without digestion, thus the methods used are potentially beneficial to biological research and clinical practice.

Directly after surgery, carcinoma and normal tissues were collected from 30 patients that had been diagnosed with thyroid cancer. This tissue was later rinsed in cold deionized water, placed in 1.5 mL cryogenic vials, and frozen in liquid nitrogen at -80 °C before analysis. 10 mg of each frozen tissue was thawed in an ice-bath, homogenized in RIPA tissue protein extraction reagent and then the tissue lysates were centrifuged at 12000 x g for 10 minutes at 4 °C in order to remove intact cells and cellular debris. Following centrifugation, the supernatant of each sample was mixed in a 1/40 ratio with the matrix. 1 μL of the final mixture was spotted on a MALDI plate and then dried in ambient temperatures before analysis was performed. The analyses were performed using a mass spectrometer that had been modified with the CovalX HM3 detection system.

The use of the CovalX HM3 system allowed for researchers to detect and analyze proteins with high molecular weights in the same tissues as those with smaller molecular weights. There were clear discrepancies found between the peaks of the carcinoma and normal tissues. Human albumin was found to create high intensities within carcinoma tissue in all 21 sample sets of carcinoma and normal tissue that were analyzed. The researchers suggest that this new method of using a high-mass detection system such as the CovalX HM3 can allow for the analysis of many different types of proteins as well as being beneficial in drug development.