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Colorectal cancer and gastric cancer are the most prevalent gastrointestinal malignancies worldwide, and early detection of these cancers is crucial to reduce their incidence and mortality. RNA methylation plays an important regulatory role in a variety of physiological activities, and it has drawn great attention in recent years. Methylated adenosine (A) modifications such as N6-methyladenosine (m6A), N1-methyladenosine (m1A), 2′-O-methyladenosine (Am), N6,2′-O-dimethyladenosine (m6Am), and N6,N6-dimethyladenosine (m62A) are typical epigenetic markers of RNA, and they are closely correlated to various diseases including cancer. Serum is a valuable source of biofluid for biomarker discovery, and determination of these adenosine modifications in human serum is desirable since they are emerging biomarkers for detection of diseases. In this work, a targeted quantitative analysis method using hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC-MS/MS) was developed and utilized to analyze these methylated adenosine modifications in serum samples. The concentration differences between the healthy volunteers and cancer patients were evaluated by Mann–Whitney test, and receiver operator characteristic (ROC) curve analysis was performed to access the potential of these nucleosides as biomarkers. We demonstrated the presence of the m6Am in human serum for the first time, and we successfully quantified the concentrations of A, m6A, m1A, and m6Am in serum samples from 99 healthy controls, 51 colorectal cancer patients, and 27 gastric cancer patients. We found that the levels of m6A and m6Am in serum were both increased in colorectal cancer or gastric cancer patients, compared to that in healthy controls. These results indicate that m6A and m6Am in serum may act as potential biomarkers for early detection and prognosis of colorectal cancer and gastric cancer. In addition, the present work will stimulate investigations on the effects of adenosine methylation on the initiation and progression of colorectal cancer and gastric cancer.

• A rapid, sensitive and reliable HILIC-MS/MS method for determination of adenosine and its modifications was developed and validated. • The method was successfully applied to quantify adenosine and its modifications in urine and plasma samples from breast cancer patients and healthy controls. • Accurate quantification was realized by using stable isotope dilution method. • Levels of m6A, A m and m6A m were elevated and m1A was reduced in the urine from breast cancer patients. • Level ratios of m6A/A, m1A/A and m6A m /A were all reduced in plasma from breast cancer patients. RNA modifications have been revealed to be essential in many biological activities, and their disorders are associated with various human diseases, including cancers. 2′-O-methyladenosine (A m), N 1-methyladenosine (m1A), N 6-methyladenosine (m6A), N 6,2′-O-dimethyladenosine (m6A m) and N 6, N 6-dimethyladenosine (m6 2 A) are important adenosine (A) modifications. The noninvasive collection of urine samples and the diverse contents of metabolites in plasma make them favored biofluids for biomarkers discovery. In this work, we established a hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) method to quantify these six nucleosides in urine and plasma of healthy controls and breast cancer (BC) patients. The limit of detection (LOD) for A, A m , m1A, m6A, m6A m , and m6 2 A were 0.0025, 0.01, 0.05, 0.005, 0.005, and 0.005 nM. The results showed that the concentrations of A m , m6A, and m6A m were increased, whereas m1A was decreased in the urine of BC patients compared with the healthy controls. We also found that the level ratios of m1A/A, m6A/A, and m6A m /A were all reduced in plasma from BC patients, compared with healthy controls. Interestingly, these ratios of methylated adenosine nucleosides to adenosine in plasma could better discriminate BC patients from healthy controls, compared to the levels of these nucleosides. The present study not only suggests these modified adenosines can act as noninvasive biomarkers of BC but also will contribute to investigating the impacts of RNA methylation on the occurrence and development of BC. [ABSTRACT FROM AUTHOR]

Colorectal cancer and gastric cancer are the most prevalent gastrointestinal malignancies worldwide, and early detection of these cancers is crucial to reduce their incidence and mortality. RNA methylation plays an important regulatory role in a variety of physiological activities, and it has drawn great attention in recent years. Methylated adenosine (A) modifications such as N 6 -methyladenosine (m 6 A), N 1 -methyladenosine (m 1 A), 2'- O -methyladenosine (Am), N 6 ,2'- O -dimethyladenosine (m 6 A) are typical epigenetic markers of RNA, and they are closely correlated to various diseases including cancer. Serum is a valuable source of biofluid for biomarker discovery, and determination of these adenosine modifications in human serum is desirable since they are emerging biomarkers for detection of diseases. In this work, a targeted quantitative analysis method using hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) was developed and utilized to analyze these methylated adenosine modifications in serum samples. The concentration differences between the healthy volunteers and cancer patients were evaluated by Mann-Whitney test, and receiver operator characteristic (ROC) curve analysis was performed to access the potential of these nucleosides as biomarkers. We demonstrated the presence of the m N 6 , N 6 -dimethyladenosine (m 6 2 A) are typical epigenetic markers of RNA, and they are closely correlated to various diseases including cancer. Serum is a valuable source of biofluid for biomarker discovery, and determination of these adenosine modifications in human serum is desirable since they are emerging biomarkers for detection of diseases. In this work, a targeted quantitative analysis method using hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) was developed and utilized to analyze these methylated adenosine modifications in serum samples. The concentration differences between the healthy volunteers and cancer patients were evaluated by Mann-Whitney test, and receiver operator characteristic (ROC) curve analysis was performed to access the potential of these nucleosides as biomarkers. We demonstrated the presence of the m 6 Am in human serum for the first time, and we successfully quantified the concentrations of A, m 6 A, m 1 A, and m 6 Am in serum samples from 99 healthy controls, 51 colorectal cancer patients, and 27 gastric cancer patients. We found that the levels of m 6 A and m 6 Am in serum were both increased in colorectal cancer or gastric cancer patients, compared to that in healthy controls. These results indicate that m 6 A and m 6 Am in serum may act as potential biomarkers for early detection and prognosis of colorectal cancer and gastric cancer. In addition, the present work will stimulate investigations on the effects of adenosine methylation on the initiation and progression of colorectal cancer and gastric cancer., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Hu, Fang, Mu, Huang, Zheng, Yuan and Guo.)