Bottom-up mass spectrometric sequencing of microRNA

Wambua, Dickson M.; Ubukata, Makoto; Dane, John; Cody, Robert B.; Chiu, Norman H. L.

doi:10.1039/c4ay01519c

Cited by 9 publications

(12 citation statements)

References 34 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“… 13 Acid hydrolysis can be performed directly in the MALDI matrix, circumventing the need for desalting after mass ladder generation. 21 Sequence confirmation for modified L-RNA has also been demonstrated using liquid chromatography (LC) coupled ESI-MS, making use of an end-labeling technique to generate single-ended reads free of internal fragments. 22 Nonetheless, no generalized procedure for fragmentation and de novo bidirectional sequencing of modified RNA has been presented.…”

Section: Introductionmentioning

confidence: 99%

Bidirectional Direct Sequencing of Noncanonical RNA by Two-Dimensional Analysis of Mass Chromatograms

et al. 2015

View full text Add to dashboard Cite

Mass spectrometry (MS) is a powerful technique for characterizing noncanonical nucleobases and other chemical modifications in small RNAs, yielding rich chemical information that is complementary to high-throughput indirect sequencing. However, mass spectra are often prohibitively complex when fragment ions are analyzed following either solution phase hydrolysis or gas phase fragmentation. For all but the simplest cases, ions arising from multiple fragmentation events, alternative fragmentation pathways, and diverse salt adducts frequently obscure desired single-cut fragment ions. Here we show that it is possible to take advantage of predictable regularities in liquid chromatographic (LC) separation of optimized RNA digests to greatly simplify the interpretation of complex MS data. A two-dimensional analysis of extracted compound chromatograms permits straightforward and robust de novo sequencing, using a novel Monte Carlo algorithm that automatically generates bidirectional paired-end reads, pinpointing the position of modified nucleotides in a sequence. We demonstrate that these advances permit routine LC-MS sequencing of RNAs containing noncanonical nucleotides, and we furthermore examine the applicability of this approach to the study of oligonucleotides containing artificial modifications as well as those commonly observed in post-transcriptionally modified RNAs.

show abstract

Section: Introductionmentioning

confidence: 99%

Bidirectional Direct Sequencing of Noncanonical RNA by Two-Dimensional Analysis of Mass Chromatograms

et al. 2015

View full text Add to dashboard Cite

show abstract

“…The algorithm searches for miRNA bearing queried partial sequence and matches it to known miRNA. Partial sequences of miR-153 that were obtained through mass spectrometric measurements in our laboratory [ 6 ] were queried in MicroRNA Multitool, and the results of matching number of miRNA are tabulated in Table 1 . From these results, it is clear that hsa-miR-153 (whose sequence is 5′ p UUGCAUAGUC ACAAAAGUGAUC-OH 3′) can be positively identified using the unique fragment [5′ pUUGCAUAGUC 3′], which is 45.4% of the miRNA sequence.…”

Section: Discussionmentioning

confidence: 99%

“…The availability of high throughput assays for the analysis of miRNA has made MS a more appealing technique for miRNA measurements. We recently demonstrated that MS can be used for the accurate detection and de novo sequencing of miRNA [ 6 ]. Nakayama et al also reported a method for the direct identification of miRNA using high resolution mass spectrometry in conjunction with database searching [ 7 ].…”

Section: Introductionmentioning

confidence: 99%

MicroRNA MultiTool: A Software for Identifying Modified and Unmodified Human microRNA Using Mass Spectrometry

Cui

Chiu

Wambua

2017

ncRNA

View full text Add to dashboard Cite

microRNA (miRNA) are short endogenous non-coding RNA that play a crucial role in post-transcriptional gene regulation and have been implicated in the initiation and progression of 160+ human diseases. Excellent analytical methods have been developed for the measurement of miRNA by mass spectrometry. However, interpretation of mass spectrometric data has been an incapacitating bottleneck in miRNA identification. This study details the development of MicroRNA MultiTool, a software for the identification of miRNA from mass spectrometric data. The software includes capabilities such as miRNA search and mass calculator, modified miRNA mass calculator, and miRNA fragment search. MicroRNA MultiTool bridges the gap between experimental data and identification of miRNA by providing a rapid means of mass spectrometric data interpretation.

show abstract

“…For sinapinic acid (SA) matrix solution, 20.0 mg of SA was dissolved in 1.0 mL of 50% ACN with 0.1% TFA. For 3-hydroxypicolinic acid (3-HPA) matrix solution, 35.0 mg of 3-HPA and 8.80 mg of ammonium citrate dibasic were dissolved in 1.0 mL of 10% ACN with 0.1% TFA [17]. To dissolve the powder of each matrix compound, the matrix solution was vortexed 1-2 min.…”

Section: Preparation Of Solutionsmentioning

confidence: 99%

“…For instance, 2,5-dihydroxybenzoic acid (DHB) is the preferred MALDI matrix for measuring protein molecules. Whereas, 3-hydroxypicolinic acid (3-HPA) is the MALDI matrix for oligonucleotide measurements [17]. To ensure the approach of using MAC ions as internal references is applicable when other MALDI matrices are used, the driftscopes of several commonly used MALDI matrices were acquired.…”

Section: Detection and Characterization Of Matrix Cluster (Mac) Ionsmentioning

confidence: 99%

MALDI matrix cluster ions as internal references for ion mobility measurements

Mwangi

Todd

Chiu

2019

Int. J. Ion Mobil. Spec.

Self Cite

View full text Add to dashboard Cite

In the absence of high vacuum, the mobility of ions in an electric field is dependent on the charge, mass, shape and size of the ions. To achieve accurate calibration for ion mobility measurements, it is important to select calibrants with comparable physical properties to the molecular ions of interest. The size of an ion is often referred as its collision cross section (CCS). Currently, the information on the CCS values of various types of molecular ions are limited, thus representing a challenge to the calibration of ion mobility spectrometry. In this study, instead of finding a way to improve the calibration of ion mobility measurements, the intrinsic by-products of the conventional matrix-assisted laser desorption/ionization (MALDI) technique, namely MALDI matrix cluster (MAC) ions, are being used as internal references for ion mobility measurements. The standard MALDI matrix and sample preparation method are used. During the MALDI ionization process, MAC ions are generated and co-exist with the molecular ions of interest within the ion source. Our results indicate the MAC ions do possess suitable ion mobility characteristics, thus allowing the MAC ions to serve as internal reference for ion mobility measurements. The MAC ions cannot be used as internal standard or calibrants for ion mobility measurements, because their molecular structures as well as their CCS are unknown. However, the detection of MAC ions can allow us to determine whether the normal operation as well as the expected performance on the sensitivity and resolution of ion mobility spectrometry are achievable and reproducible. The MAC ions can also facilitate the transfer of specific experimental protocols between ion mobility instruments and/or laboratories. No extra materials, equipment or procedure are required for using MAC ions. For the proof of concept, all experimental work in this study was carried out on a traveling wave ion mobility mass spectrometry platform.

show abstract

Bottom-up mass spectrometric sequencing of microRNA

Cited by 9 publications

References 34 publications

Bidirectional Direct Sequencing of Noncanonical RNA by Two-Dimensional Analysis of Mass Chromatograms

Bidirectional Direct Sequencing of Noncanonical RNA by Two-Dimensional Analysis of Mass Chromatograms

MicroRNA MultiTool: A Software for Identifying Modified and Unmodified Human microRNA Using Mass Spectrometry

MALDI matrix cluster ions as internal references for ion mobility measurements

Contact Info

Product

Resources

About