A Novel Multiplex-Based, Nested Data-Independent Acquisition Method

Data-independent acquisition (DIA) is a method for structure determination whereby all ions within a designated m/z range are fragmented and analyzed in a second stage of tandem mass spectrometry (MS/MS).

DIA is a potentially useful technique in various fields of –omics, forensics and environmental analysis. However, current MS technology is not capable of fulfilling the expectations of providing comprehensive MS/MS data while also following the elution profile of chromatographic peaks.

Kaufmann and Walker present a new “nested DIA” approach, a multiplex-based method, to more closely achieve this objective.¹ The method represents a promising strategy toward comprehensive MS/MS, although it still has some limitations that prevent it from being used routinely.

Conventional DIA is most commonly performed using time-of-flight (TOF) instruments. Currently available DIA deals with mass isolation windows that are clearly wider than unit mass, which is a problem when it comes to interfering peaks. Certain methods exist that allow smaller windows to be used, but very narrow mass windows lead to a small number of summed TOF transients within a spectrum. This in turn leads to poor signal-to-noise ratios.

Instead of a quadrupole-TOF instrument, the team used a Q Exactive Plus mass spectrometer (Thermo Scientific) in positive electrospray ionization mode coupled to a liquid chromatography system. The nested DIA scans were programmed using the application programming interface (API) software (Thermo Scientific).

The team looked at a standard sample of bovine muscle tissue. The combination of a conventional DIA and a binary tree multiplexed scan resulted in a nested DIA scan. The instrument first acquired a full scan (m/z = 140–1,000) without applying any collision energy. This was followed by eight DIA scans covering the mass range 140–1,000. Four nested multiplexed scans followed the conventional DIA scans. The whole customized scan sequence comprised 12 subscans. The setup permitted the evaluation of calculated mass window widths corresponding to 13.4 Da. The chosen settings resulted in a cycle time of 1.31 s.

The research team extracted all 12 acquired scans corresponding to the retention time of the analytes, and then imported the accurate masses and signal abundances into a spreadsheet. Following this, they aligned the product ion accurate masses across the 12 spectra and used this information to assign each particular product ion to a precursor mass isolation window.

Quadrupole-orbitrap instruments are able to perform multiplexed data acquisition, enabling Kaufmann and Walker to conceive their new nested DIA approach.

The API software also helped the team achieve multiplexing, as the software permits application-specific programming of customized scans. The programmed scans use the quadrupole to extract a number of mass range segments out of a full scan. The ions originating from the mass range segments were stored in the curved linear trap and injected as a whole into the Orbitrap mass analyzer.

The approach used differs from other published multiplexed DIA approaches in that systematically nested mass range segments were used to obtain narrow extraction windows using the least possible number of scans. The addition of a single scan halves the width of the MS/MS isolation window.

Compared to conventional DIA, the nested method only needed half the number of scans to obtain the same isolation width improvement. It also had shorter cycle times. Overall, nested DIA allowed narrower mass isolation windows and produced cleaner product ion spectra.

Reference
1. Kaufmann, A., and Walker, S. (2016) “Nested data independent MS/MS acquisition,” Analytical and Bioanalytical Chemistry, 408(18) (pp. 5031–5040), doi: 10.1007/s00216-016-9607-8.