Page 17 - Oligonucleotide Therapeutics Solution Guide

Characteristic analysis
Quality Control

Sequence Confirmation & Molecular Weight Confirmation LCMS-9030 / MALDI-8030

Features
Analysis of Oligonucleotide Therapeutics
The LCMS-9030 is a Q-TOF type mass spectrometer with two types of ion mass separation mechanisms: quadrupole and time-of-flight. While high- Modification
using MALDI-8030 and LCMS-9030 resolution and high-precision ESI mass spectrometers enable precise mass measurement of oligonucleotides, routine oligonucleotide sequence Target selection
analysis using ESI-MS is still difficult, and complete internal oligo-sequences are rarely obtained using these general ESI-MS/MS techniques. The
click here reason for this is that the Collision-induced dissociation (CID) used in ESI-MS/MS has difficulty in obtaining fragment ions that are effective for the
internal sequence assignment of oligonucleotides.
1)
On the other hand, in-source decay (ISD) using MALDI-TOF MS was reported as a useful method to conduct sequence analysis , although the
instruments often employed do not have sufficient specification for exact mass measurement.
• Confident characterization of oligonucleotides
Reference
• Elemental formula confirmation by ESI-QTOF 1) Shimizu H, Jinno F, Morohashi A, Yamazaki Y, Yamada M, Kondo T, Asahi S. J Mass Spectrom.
2012 Aug;47(8):1015-22.
benefits • Information of sequence by MALDI-ISD
New TOF Technologies UFgrating (Pat. US 10020181) Unprotected Excision Oligomer synthesis
Methods and Results -5 Ion accumulation in the collision cell,
1342.1297 UFgrating (Pat. US 10020181) synchronized perfectly with short cycles of data
ESI mass spectrum
8.0e4 acquisition, maximizes sensitivity.
Sample Phosphorothioated oligonucleotides, differing in the structure Deconvoluted spectrum 6.0e4 1.80e5 Shimadzu’s world-class manufacturing capability
-4
-6
of the sugar constituents (Figure 1) 1.8e5 6714.6792 6715.6719 6716.6738 4.0e4 1118.2749 1677.9125 has enabled the ion acceleration electrode to be
LNA-Oligo (m/z 6711.6731) S-Oligo (m/z 6431.7240) 1.6e5 2.0e4 1364.4985 1691.8940 made with substantial mechanical strength. This UF-FlightTube (Patent Pending)
1.4e5 6717.6802 512.9966 958.3779 1376.6822 1705.8738 2237.5522 2897.1761
Conc., Volume 10 pmol/µL, 1 µL 1.2e5 6713.6768 0.0e0 500 1000 1500 2000 2500 grating is able to withstand the high voltages With excellent architecture, the UF-FlightTube
m/z needed for ultrafast ion pulsing. prevents and withstands subtle deformations
Preparation Dilution in ultrapure water to the concentrations above. 1.0e5 6718.6816 caused by temperature changes, affording
8.0e4
6.0e4 6712.6768 6719.6802 stability of performance.
Molecular An exact mass measurement in negative ion detection was 4.0e4 [M] 6720.6807 Traditional mesh electrodes for ion
weight conducted using a LCMS-9030. 2.0e4 6711.6733 6721.6821 6722.6753 transmission lack mechanical strength, iRefTOF (Pat. US 8772708, 9490114)
confirmation The solvent consisted of 50 mmol/L HFIP, 10 mmol/L DIPEA, and 0.0e0 6708 6710 6712 Fig. 1 Sequences ofoligonucleotides 6720 6722 6724 6726 質量 limiting acceleration voltage.
6716
6714
6718

acetonitrile, was applied to the ESI at a flow rate of 0.2 mL/min. Figure 2 Exact mass measurement of LNA-Oligo A computationally ideal electrostatic eld has
The MS range of the QTOF was set as m/z 500 to 3000. become a reality. Meticulously manufactured Purification
Deconvolution of ESI spectra was performed with ReSpect in Shimadzu’ s UFgrating has superior plate electrodes are stacked to create a re ectron
LabSolutions Insight. [M-2H] - mechanical strength over that compensates for the energy distribution of
conventional electrodes.
4-
Results Multiply-charged ions of the oligos distributed from [M-4H] to This unique grating structure makes it ions with no compromise in either resolution or
[M-6H] were observed (Figure 2, inset). Exact masses of two possible to apply a higher voltage. sensitivity.
6-
oligos were successfully obtained by deconvolution of the ESI
spectra. m/z 6711.6733 was obtained from the ESI-MS of the [M-H] -
LNA-Oligo (Figure 2), and m/z 6431.7241 from the S-oligo.
w 6
w 5
Internal ISD with negative ion detection was performed on a w 11 Key Technology
w 2 w 7 w 12
sequence MALDI-8030, dual polarity benchtop linear MALDI-TOF MS. w 8 w 10 w 13
information 3-HPA (3-hydroxypicolinic acid) and ammonium citrate were w 3 w 4 w 9 w 14 w 15 The LCMS-9030 uses newly patented technologies to deliver both high resolution and
analysis applied to MALDI-ISD measurement as matrix and additives, w 16 w 17 w 18 w 19
respectively. Matrix solution and samples were layered on a accurate mass, attributes essential for confident formula assignment and unknown
stainless MALDI plate. A transition from MS measurement to ISD identification. The high-efficiency ion guides, quadrupole, and collision cell enable
in the instrument is quick and easy by simply increasing laser high sensitivity for the detection of trace-level compounds. Unique UFgrating and
irradiation power. LCMS-9030 Quality Control
iRefTOF technologies ensure ultrafast acceleration of ions into the flight tube (UF-
Results MALDI-ISD ladder sequence confirmation of the LNA-Oligo was Characteristic analysis
shown in Figure 3. FlightTube) and ideal reflection of those ions back to the detector. The result is high-
Fragment ions, denoted as a- and w-seiries ions, were assigned Figure 3 ISD of LNA-Oligo, and assignment of a- and w-series ions. speed data acquisition compatible with the high-throughput laboratory.
by matching against the theoretical average masses. w-ions
derived from almost the whole oligo sequences were found in
the spectra. Only one or two ions derived from the 3’-terminal
units were missed due to an overlap with the matrix signals.
In the case of the S-oligo, with the exception of two 5’-terminal
units, almost a complete series of a-ions were detected. However,
in the case of LNA-oligo, the detected a-ions corresponded to Internal Sequence Analysis by
those derived from the internal sequence, indicating that a-ions
support confirmation of the sequence. In-source Decay (ISD) DDS
The ISD cleavage shown in the figure below enables easy sequence analysis even for
MALDI-TOF MS without MS/MS capability. Pharmacokinetics
Cleavage site by ISD
Figure 1 Sequences of oligonucleotides
Conclusions Intensity (%)
Other
Exact mass measurements using the LCMS-9030 resulted in doubtless consistency between the theoretical and observed masses. ISD
using the MALDI-8030 resulted in mainly an internal sequence information, which is difficult to obtain with any MS/MS technique. The
MALDI-ISD and ESI-QTOF are a useful combination to characterize oligonucleotide therapeutics.
MALDI-8030
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