PulseDIA: in-depth data independent acquisition mass spectrometry using enhanced gas phase fractionation

Abstract

An inherent bottleneck of data independent acquisition (DIA) analysis by Orbitrap-based mass spectrometers is the relatively large window width due to the relatively slow scanning rate compared to TOF. Here we present a novel gas phase separation and MS acquisition method called PulseDIA-MS, which improves the specificity and sensitivity of Orbitrap-based DIA analysis. This is achieved by dividing the ordinary DIA-MS analysis covering the entire mass range into multiple injections for DIA-MS analyses with complementary windows. Using standard HeLa digests, the PulseDIA method identified 61,078 peptides from 7,308 protein groups with ten MS injections of 30 min LC gradient. The PulseDIA scheme containing two complementary windows led to the highest gain of peptide and protein identifications per time unit compared to the conventional 30 min DIA method. We further applied the method to profile the proteome of 18 cholangiocarcinoma (CCA) tissue samples (benign and malignant) from nine patients. PulseDIA identified 6,063 protein groups in these CCA samples, with 23% increase of protein identifications, compared to the classical DIA method. The missing value for protein matrix dropped by 25% with PulseDIA acquisition. 618 proteins were significantly dysregulated in tumorous CCA samples. Together, we presented and benchmarked an alternative DIA method with higher sensitivity and lower missing rate.

Accelerated Lysis and Proteolytic Digestion of Biopsy-Level Fresh-Frozen and FFPE Tissue Samples Using Pressure Cycling Technology

“…12 Two microliters of peptides (0.25 μg/μL) were separated at a flow rate of 300 nL/min along a 45 min 3−28% linear LC gradient (buffer A: 2% ACN, 0.1% FA in MS grade water; buffer B: 98% ACN, 0.1% FA in MS grade water) on a precolumn (3 μm, 100 Å, 20 mm × 75 μm, Thermo Fisher Scientific), followed by an analytical column (1.9 μm, 100 Å, 150 mm × 75 μm), using the Ultimate 3000 nanoLC-MS/MS system (Dionex LC-Packings) coupled with a QEHF-X (Thermo Fisher Scientific). 12 The PulseDIA was performed over a m/z ranging from 390 to 1210 at a resolution of 60 000 (at m/z 200) in an Orbitrap using an AGC target value of 3E6 charges and ion funnel RF settings of 40. The PulseDIA method was performed with four injections, and the fixed isolation window was used as described previously.…”

“…The PulseDIA method was performed with four injections, and the fixed isolation window was used as described previously. 12 The MS2 spectra were acquired at a resolution of 30 000 (at m/z 200) in the Orbitrap using an AGC target value of 1E6 and max IT of 50 ms.…”

“…PulseDIA data files were converted into mzML using Msconvert (v3.0.19172-57d620127) and analyzed using DIA-NN (v 1.7.0) 14 based on the protocol described previously. 12 The DIA Pan-Human Library (DPHL) was used as the spectral library for PulseDIA target extraction analysis, with 14 786 protein groups and 396 245 transitions. 15 Briefly, the peptide length range was 7−30, the precursor m/z range was 400−1200, and the product ion m/z range was 100−1500.…”

Data‐Independent Acquisition Mass Spectrometry‐Based Proteomics and Software Tools: A Glimpse in 2020

“…Various schemes of DIA strategies have been implemented since the beginning of this century, including but not limited to Shotgun CID, [9] DIA, [5] MS E , [10] PAcIFIC, [11] AIF, [12] SWATH-MS, [6] MSX, [13] SONAR, [14] WiSIM, [15] BoxCar DIA, [16] Scanning SWATH, [17] diaPASEF, [18] and PulseDIA [19] (Figure 1, Table 1), but not all schemes have been widely revisited. In Shotgun collision-induced dissociation (Shotgun CID) [9] induces in-source fragmentations where all ions co-eluting from chromatographic separation into the mass spectrometer are fragmented in parallel.…”

“…The data acquisition method BoxCar [16] increasing the MS1 dynamic range is also available on DIA. PulseDIA [19] is an iterative gas phase fractionation method in which replicate sample injections are analyzed using differentially segmented DIA-MS methods in order to decrease window width and improve specificity and sensitivity, enabling nearly equal distribution of the proteome into any number of fractions. Recently parallel accumulation-serial fragmentation combined with data-independent acquisition (diaPASEF) was developed to utilize the ion mobility of peptide precursor in a trapped ion mobility mass spectrometer (TIMS) that can fully sample peptide precursor ion current Fangfei Zhang obtained her master's degree in Bioinformatics from ETH Zurich in March 2019 supervised by Prof. Dr. Ruedi Aebersold focusing on the histone posttranslational modifications using data-independent acquisition (DIA).…”