Multiplexed MS/MS for improved data-independent acquisition

Abstract

In mass spectrometry-based proteomics, data-independent acquisition (DIA) strategies can acquire a single data set useful for both identification and quantification of detectable peptides in a complex mixture. However, DIA data are noisy owing to a typical five- to tenfold reduction in precursor selectivity compared to data obtained with data-dependent acquisition or selected reaction monitoring. We demonstrate a multiplexing strategy, MSX, for DIA analysis that increases precursor selectivity fivefold.

Figure 1. Multiplexed Data Independent Acquisition (MSX)

A common implementation of data independent acquisition (DIA) is to use a repeated cycle of wide isolation window MS/MS scans to cover a mass range. In this example, the 500–900 m/z range is covered with 40 scans each sampling a single 10 m/z wide window. In multiplexed DIA (MSX), each scan isolates five 4 m/z wide windows prior to fragment ion mass analysis. The five windows isolated in each scan are chosen randomly from the set of 100 possible non-overlapping windows covering the 500–900 m/z range. Each mixed MSX spectrum is de-multiplexed into the five component spectra corresponding to each isolated window.

Figure 2. Demultiplexing Reduces Chemical Noise and Improves Selectivity

The full b- and y- ion series for the peptide GPLVLEYETYR are plotted from an MSX experiment on the soluble fraction of S. cerevisiae lysate prior to (a, c) and after (b, d) demultiplexing. Prior to demultiplexing, there are many other peaks of similar or greater intensity than the peak for GPLVLEYETYR (indicated with an arrow in (a)). After demultiplexing, peaks from other precursors are effectively removed and the true peak is by far the most intense (b). Additionally, the demultiplexed peak (d) contains far less interference than the unprocessed peak (c).

Figure 3. Quantitation of the LVNELTEFAK Peptide by MSX and MS1

A commercial six protein digest was spiked into a complex matrix (soluble S. cerevisiae lysate) at amounts ranging from 50 attomoles – 100 femtomoles on column. MSX data were acquired with an MS1 scan interleaved every ten scans. The signal intensity for each spike in point (normalized to two background peptides) for the peptide LVNELTEFAK++ is plotted in (a) with (right pane) and without (left pane) log scaling of the x and y axes. The lower limit of detection is 0.41 femtomoles and 1.02 femtomoles for MSX and MS1-based quantitation respectively. The slopes of the regression lines were 0.030 ± 0.004 (95% confidence interval) and 0.071 ± 0.002 for MSX and MS1 respectively. (b) and (c) show the MS1 and MSX signal at 1.02 femtomoles. Although on average MS1 quantitation is more sensitive than MSX, chemical noise hinders quantitation of this peptide by MS1 and MSX is more sensitive due to greater selectivity.