Supporting Information. Scanning Quadrupole Data Independent Acquisition Part A Qualitative and Quantitative Characterization

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1 S Supporting Information Scanning Quadrupole Data Independent Acquisition Part A Qualitative and Quantitative Characterization M. Arthur Moseley, Christopher J. Hughes 2, Praveen R. Juvvadi 3, Erik J. Soderblom, Sarah Lennon 2, Simon R. Perkins 4, J. Will Thompson, William J. Steinbach 3,5, Scott J. Geromanos 6, Jason Wildgoose 2, James I. Langridge 2, Keith Richardson 2, Johannes P.C. Vissers 2. Proteomics and Metabolomics Shared Resource Center for Genomic and Computational Biology, Duke University Medical Center, Durham, NC 2. Waters Corporation, Wilmslow, United Kingdom 3. Division of Pediatric Infectious Diseases, Department of Pediatrics, Duke University Medical Center, Durham, NC 4. Institute of Integrative Biology, University of Liverpool, Liverpool, United Kingdom 5. Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC 6. Waters Corporation, Milford, MA Page S2 / Table S Page S3 / Figure S Page S4 / Figure S2 Page S5 / Figure S3 Page S6 / Figure S4 Page S7 / Figure S5 Scanning quadrupole DIA and DDA processing and search parameters. Limited HeLa load scanning quadrupole DIA LCMS data analysis. Highest observed identification rate from untargeted search results per experiment and sample type, optimizing scanning quadrupole DIA acquisition parameters and gradient lengths. Log 2 relative profile abundance profiles scanning quadrupole DIA. Targeted library search identification example. Quantitative precision summary of scanning quadrupole DIA analysis of HeLa human cell line sample showing the correlation, Hi(n) response vs. amount injected, as a function of dynamic range.

2 S2 Supplementary Table. Scanning quadrupole DIA and DDA processing and search parameters. processing parameters chromatographic peak width (FWHM) resolution lock mass (m/z) lock mass window (m/z) low enegery (MS) threshold high energy (MS2) threshold mzml conversion scanning quadurpole DIA DDA automatic automatic default settings untargeted search parameters precursor tolerance fragment tolerance number of fragment ions fixed modification variable modification digest missed cleavages protein FDR (%) peptide FDR (%) targeted search parameters match tolerance retention time tolerance automatic (± 3.5 ppm)* automatic (± 9.0 ppm)* 5 Carbamidomethyl (C) Oxidation (M) trypsin ± 0 ppm ± min ± 20 ppm ± 0. Da Carbamidomethyl (C) Oxidation (M), Acetyl (protein Nterm) trypsin as specified in PXD0044; MSConvert ( * set by resolution (~ 27,500 FWHM for both MS and MS2); = not applicable. ProteinLynx Global Server v3.0.2 and IsoQuant were used for the untargeted processing and searching of the scanning quadrupole DIA data, MSconvert v2.and Mascot v2.5. for DDA data processing and searching, respectively, and development software [5,6] for the targeted searches.

3 S3 Supplementary Figure. Limited HeLa load (250 ng) LCMS data analysis showing (A) 30 s chromatographic 2D quadrupole m/z vs. TOF m/z data/distribution, (B) peak detected product ions in 5 consecutive average scanning quadrupole position bins (~ 50% of the transmission window), and (C) MS2 product ions associated with a single peak detected feature.

4 S4 Supplementary Figure 2. Highest observed identification rate from untargeted search results per experiment and sample type, optimizing scanning quadrupole DIA acquisition parameters and gradient lengths. Average (n = 3) identification values are shown with an average technical variation across all experiments smaller than 5%. 3 (D) represents gradient time, scan time, quadrupole isolation width, and loading amount oncolumn optimized results. In the instance of 3 (A), (B), and (C) (undepleted human plasma and human cell line), the amount loaded oncolumn was not optimized, but at constant value. The investigated DIA acquisition parameter and gradient length values for which the highest identification rate for each of the experiment was observed are appended to the experiment label.

5 S5 Supplementary Figure 3. Log 2 relative profile abundances Alcohol Dehydrogenase P00330 (squares), Glycogen Phosporylase P00489 (circles), Enolase P00924 (diamonds)*, Enolase 2 P00925 (triangles)**, and Bovine Serum Albumin P02769 (reversed triangles). [A] concentration = 2[B] concentration. The error bars represent protein abundance variance. Dashed line = expected profile value; * = protein selected for normalization; ** = contaminant; = exceeding expected level (caused by a reduced number of identified peptides due to the lower Mw (smaller number of detectable peptides) and lower amount injected oncolumn).

6 S6 Supplementary Figure 4. Targeted library search identification example of YLEGTSCIAGVLVPAK showing the scanning quadrupole DIA query spectrum (top) and the DDA library spectrum (bottom). Ion fragment type annotation = y (red), b (blue), NH 3 or H 2 O neutral loss (green), library and/or sequence matched (black) and not identified (grey).

7 S7 Supplementary Figure 5. Graphical quantitative precision summary of scanning quadrupole DIA analysis of HeLa human cell line sample showing the correlation, Hi(n) response vs. amount injected, as a function of dynamic range (µmol target protein/mol total protein).