Scanning Quadrupole Data-Independent Acquisition, Part A: Qualitative and Quantitative Characterization

Abstract

A novel data-independent acquisition (DIA) method incorporating a scanning quadrupole in front of a collision cell and orthogonal acceleration time-of-flight mass analyzer is described. The method has been characterized for the qualitative and quantitative label-free proteomic analysis of complex biological samples. The principle of the scanning quadrupole DIA method is discussed, and analytical instrument characteristics, such as the quadrupole transmission width, scan/integration time, and chromatographic separation, have been optimized in relation to sample complexity for a number of different model proteomes of varying complexity and dynamic range including human plasma, cell lines, and bacteria. In addition, the technological merits over existing DIA approaches are described and contrasted. The qualitative and semiquantitative performance of the method is illustrated for the analysis of relatively simple protein digest mixtures and a well-characterized human cell line sample using untargeted and targeted search strategies. Finally, the results from a human cell line were compared against publicly available data that used similar chromatographic conditions but were acquired with DDA technology and alternative mass analyzer systems. Qualitative comparison showed excellent concordance of results with >90% overlap of the detected proteins.

Keywords: data-independent acquisition; label-free quantitation; scanning quadrupole.

Figure 1.

(A) MS instrument optics/configuration; shown inset is the transmission profile as a function of time. (B) Quadruple collision energy profiles as a function of experiment type and time (and average quadrupole position in bins). (C) Nested 2D MS (quadrupole m/z vs TOF m/z) MS1 and MS2 data sets, including typical quadrupole transmission profiles for fragments of DFNVGGYIQAVLDR (PYGM_RABIT) eluting at 23.7 min. (D) Quadrupole extracted TOF MS1 and MS2 spectra for the peptide shown in panel C following 2D peak (precursor m/z and fragment m/z) detection of the scanning quadrupole DIA data. The extraction width used in this case was ∼10% of the quadrupole peak width.

Figure 2.

2D m/z quadrupole versus m/z TOF distributions and reconstructed spectra, showing the aggregate of all average quadrupole positions and a single average quadrupole position 2D MS1 and MS2 distributions, (A) and (B), respectively, and aggregate and single average quadrupole position MS1 and MS2 spectra, (C) and (D), respectively, for SADTLWGIQK (LDHA_HUMAN) eluting at 61.7 min.

Figure 3.

Scanning quadrupole DIA acquisition parameter and gradient optimization examples for the normalized number of identified protein groups for (A) E. coli, squares = 30 min gradient and circles = 45 min gradient; (B) human cell line, squares = 30 min gradient and circles = 45 min gradient; (C) squares, human cell line; circles, human undepleted plasma; and (D) human cell line, squares = 0.3 s scan time and circles = 0.5 s scan time. Average (n = 3) relative identification values are shown with an average technical variation across all experiments smaller than 5%.

Figure 4.

Graphical qualitative identification summary of scanning quadrupole DIA analysis of HeLa human cell line sample. Left to right and clockwise: Amino acid sequence coverage as a function of dynamic range (μmol/mol) (A), number of peptides as a function of dynamic range (μmol/mol) (B), quantitative response (normalized slope ((μmol/mol)/amount) vs amount) as a function of dynamic range (μmol/mol) (C), shown inset are median values, DIA product ion spectrum (D), Venn distribution protein identifications (gray, common; black, condition or condition pair unique) (E), product ion extracted chromatograms (F), and product ion scanning quadrupole profiles (G).

Figure 5.

Qualitative comparison scanning quadrupole DIA (unfractionated) results versus library DDA (concatenated (96/12) off-line 2D basic pH reversed-phase fractionated and identification replication filtered) and public DDA (6 fraction off-line 2D basic pH reversed phase fractionated) HeLa LC–MS data. (A) Proteins and protein groups in parentheses and (B) number of identified peptides and amino sequence coverage (black, scanning quadrupole DIA; dark gray, reference DDA data; light gray, DDA library).