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RAPID QUALITATIVE AND ABSOLUTE QUANTIFICATION OF PLASMA BASED PROTEINS USING A NOVEL SCANNING QUADRUPOLE DIA ACQUISITION METHOD

Christopher J. Hughes1, Sarah Lennon

1, Lee A. Gethings

1, David Heywood

1, Florian Marty

2, Sebastian Mϋller

2, Lukas Reiter

2, Robert S. Plumb

3

1Waters Corporation, Wilmslow, UK;

2Biognosys AG, Schlieren, Switzerland;

3Waters Corporation, Milford, MA

INTRODUCTION

Quantitative proteomics often incorporates the use of stable

isotope labels (SILs) to provide absolute quantification. Recent

advancements have seen the introduction of peptide panels

allowing the quantification of over 500 proteins in plasma sample

sets. However, this is technically challenging when attempting to

acquire the data using more traditional MS acquisition modes such

as multiple reaction monitoring (MRM), since the duty cycle of the

instrument is compromised and therefore results in under

sampling. An alternative approach is to apply a data independent

analysis (DIA) methodology, allowing for high throughput whilst

also ensuring high rates of data acquisition and specificity. Here

we describe the use of a kit consisting of SIL’s capable of

quantifying >500 plasma-based proteins in conjunction with a

novel scanning quadrupole DIA acquisition schema (SONAR) in

order to quantify proteins of interest for patient cohorts diagnosed

with respiratory disorders.

METHODS Sample Preparation

Undepleted human plasma (Innovative Research, MI) originating from controls (n=6) and patients diagnosed with COPD (n=6) and asthma (n=6) were reduced, alkylated and tryptically digested overnight. Prior to LC-MS analysis, samples corresponding to their individual groups were pooled and spiked with PQ500 SIL peptides (Biognosys AG, Schlieren, Switzerland).

LC Conditions

LC system: M-class ACQUITY system Column(s): 1.8 μm CSH C18 300 μm x 100 mm NanoEase

analytical column Column temperature: 35 °C Flow rate: 7 µL/min Mobile phase: water (0.1% formic acid) (A) and acetonitrile

(0.1% formic acid) (B) Gradient: 3% to 40% B in 15 or 45 min Injection volume: 5 μL (5 μg)

MS Conditions

MS system: Xevo G2-XS QTof Ionization mode: ESI (+) at 2.2 kV Cone voltage: 30 V Acquisition mode: SONAR Acquisition range: 50 to 2000 m/z both functions (low and elevated

energy) Acquisition rate: 0.5 s both functions (low and elevated energy) Quadrupole scan range: 330 to 1100 m/z Isolation window: 20 Da Collision energy: 6 eV (low energy function) and from 14 eV to 36

eV (elevated energy function) Resolution: 30,000 FWHM

Informatics

LC-MS data were processed with Spectronaut Pulsar X (Biognosys AG, Schlieren, Switzerland). Data were searched against a human plasma specific spectral library, generated from SONAR processed data with Progenesis QI for Proteomics. Additional data visualization and statistical analysis was performed using Metaboanalyst

1.

RESULTS

The principle of SONAR is illustrated in Figure 1. This DIA methodology acquires datasets alternately with low (MS1) and elevated (MS2) collision energies

2,3. During each low and elevated energy segment, the quadrupole isolation window is scanned linearly between two user-selected positions

and 200 TOF spectra are acquired. The workflow outlining the spectral library generation and processing with Spectronaut is detailed in Figure 2.

Data were collected for undepleted plasma which was separated over either 45 or 15 minute gradients, resulting in 347 and 218 protein identifications respectively. Regardless of the gradient conditions employed, statistical analysis showed clear separation between the three cohorts, with all three technical replicates clustering together. Likewise, similar levels of protein expression were also generated when reviewing protein abundance using hierarchial clustering (Figure 3). The importance of peak sampling frequency and its effect on quantitative precision has been previously described.

2

Figure 4 shows the increased specificity provided by SONAR, resulting in non-interfered detection and extraction of MS2 fragment ions allowing proteins to be quantified over a wide dynamic range (Figure 5). Quantitative variance in terms of average %CV within each cohort (over technical replicates) was demonstrated to be 6% for both 45 and 15 minute gradients (Figure 6).

Assessing the differentiation regulation across the three cohorts, highlighted a number of key proteins to be expressed for COPD and asthma. Using the corresponding PQ500 SILs allowed for absolute quantitation to be derived for these proteins of interest (Figure 7).

Figure 1. A schematic of the SONAR acquisition method showing the behavior of the collision energy, which alternates between a constant low value and a linear ramp to generate informative precursor and frag-mentation data, respectively, for as many peptide precursor ions as pos-sible. The quadrupole scans with the same parameters in both the low and high energy CID scans.

Figure 3. Principal component analysis (PCA) showing clear separation between controls, COPD and asthma patient groups for 45 min (a) and 15 min (b) gradients. Hierarchial clustering based on Euclidean distances provide representative heatmaps, highlighting differences in protein abundances between the three cohorts for both gradient conditions (c,d).

CONCLUSION High throughput proteomics providing qualitative and highly precise quantitative data.

Absolute protein quantitation achieved using SONAR/PQ500 kit with average CV’s of 6%.

Quantification of the plasma proteome is achieved over a wide dynamic range (>4 orders).

Applicable solution which could be routinely implemented into a clinical proteomics research workflow.

Figure 2. Data analysis workflow. Spectral library generation via Progenesis QI for Proteomics is followed by data analysis and quantitative results using Spectronaut.

Figure 4. Selectivity benefits increase quantitative precision using SONAR. MS1 data representing a fi-bronectin related peptide, shows potential interfer-ence from co-eluting peptides (upper). Increased SO-NAR specificity provides a clean, non-interfered MS2 spectrum (lower).

Figure 5. Normalised abundance profile of all identified proteins from all samples/conditions. Com-bined protein numbers show >500 proteins identified and spanning a dynamic range >4 or-ders in magnitude.

Figure 7. Box-whisker plots representing a selection of proteins from the PQ500 kit over all three conditions (A). Absolute quantitation re-gression plot (B) of identified plasma proteins, details protein concen-trations corresponding to the PQ500 SILs (red box-whisker) in addition to those proteins which are identified external to the PQ500 SILs (grey box-whisker).

Figure 6. Co-efficient of variance (CV) for MS2 based quantitation. All three cohorts demonstrate a %CV of 6% for analyses over both 45 and 15 min gradients. Acquir-ing the data over a shorter gradi-ent also appears to reduce the spread of CV, maintaining CV’s for all measurements <20%

References

1. Chong J et al. MetaboAnlyst 4.0: towards more transparent and integrative metabolomics analysis. Nucl. Acids Res. (doi: 10.1093/nar/

gky310).

2. Gethings LA et al. Lipid profiling of complex biological mixtures by liquid chromatography/mass spectrometry using a novel scanning quadrupole data-independent acquisition strategy. Rapid Commun Mass Spectrom. 2017 Oct 15;31(19):1599-1606. doi: 10.1002/

rcm.7941.

3. Moseley AM et al. Scanning Quadrupole Data Independent Acquisition – Part A. Qualitative and Quantitative Characterization, J Proteome

Res. 2017 Sep 13. doi: 10.1021/acs.jproteome.7b00464.