AI-64310-CRFT

Real-Time Qualitative and Quantitative Global Proteomics Profiling Using a Hybrid Data Acquisition Scheme

Overview

Purpose:

Demonstrate a more comprehensive data acquisition scheme for performing

global protein/peptide qualitative and quantitative analysis. Evaluate data acquisition

performance by comparing to previously published methods.

Methods:

Data acquisition strategy based on HR/AM MS used for quantitative analysis

with looped narrow precursor DIA events for sequence confirmation. Utilize

consolidated spectral library information facilitating real-time data analysis to create a

targeted peptide list used for analysis across all technical replicates.

Results:

The pSMART acquisition method resulted in more peptides identified and

quantified than the standard DIA method with significantly fewer decoy matches

resulting in greater quantitation in much less time.

Introduction

The trend in proteomics is to perform global qualitative and quantitative sample

analysis without targeted precursor inclusion lists or from MS-driven MS/MS

acquisition. The goal is to sample the greatest number of peptides across the gradient

enabling global determinations across biological samples which can then determine

subsequent targeted, high-throughput experiments. Data independent acquisition

(DIA) strategies have become common due to opportunity to archive and interrogate

data using any user-defined protein/peptide lists. To increase the sampling and

characterization capabilities for global qual/quan experiments, we utilize high

resolution/accurate mass (HR/AM) MS and narrow, asymmetric DIA windows as

opposed to only DIA data. The decoupling of data sets used for quantitative and

qualitative analysis increases quality of both sets. Success is predicated on leveraging

high resolution (>70,000 per precursor target) and high mass accuracy to increase

selectivity for robust MS quantitation. In addition, the acquisition strategy enables

product ion data quality to be significantly increased because only one high quality

product ion spectrum is needed for verification. The narrow precursor isolation using

high ion accumulation times and higher resolution settings have shown greater

sensitivity and selectivity compared to standard DIA data on the same samples.

Methods

Sample Preparation

All experiments were performed using a donor sample of human plasma collected

under IRB approved protocols and stored in an EDTA stabilized tube (Becton

Dickinson, Franklin Lakes, NJ). A stock solution of human plasma was prepared

without depletion using standard trypsin digestion protocols following reduction and

alkylation. The concentration of the final stock solution was estimated to be 4 mg/µL,

divided into aliquots of 100 µL of 100 ng/µL and frozen until used. Before MS analysis,

the sample was spiked with Peptide Retention Time Calibration (PRTC) peptides

(Thermo Fisher Scientific, Rockford, IL) to a final concentration of 20 fmol on column.

A total of 1 µL was injected on column per experiment.

Liquid Chromatography

All chromatographic separation was performed using a Thermo Scientific

EASY-

nLCII

LC system with a binary solvent system of (A) 0.2% formic acid in water and(B)

0.2% formic acid in acetonitrile. Samples were loaded onto a 120 x 0.15 mm trapping

column packed with 5 µm PS-dvb particles (Polymer Labs) and the analytical

separation was performed using a 500 x 0.1 mm column packed with C18 Aq

(Bischoff). The samples were eluted from the column with a linear gradient from 5 to

45% B in 180 minutes prior to ramping to 90% B for column regeneration.

Mass Spectrometry

Thermo Scientific

Q Exactive

mass spectrometer was used for all experiments.

Two different experiments were performed, standard DIA and peptide-based.

Staggered MS and M/MS acquisition Across Retention Time (pSMART) for data

analysis. Standard DIA acquisition was performed using 25 Da precursor isolation

covering m/z 400-1200 in 32 scan events, 100 msec max ion fill times, 1e6 AGC

settings, and 35,000 resolution (@m/z 200). The pSMART acquisition settings for MS

was 5e6 AGC setting and 140,0000 resolution (@m/z 200) and DIA events were

independently acquired using 5 Da precursor isolation for a precursor range of m/z

400-800, 10 Da for m/z 800-1000, and 20 Da for m/z 1000-1200. Each narrow DIA

was acquired using 150 msec max ion fill times, 1e6 AGC settings, and 35,000

resolution (@m/z 200). A custom acquisition script was used to perform real-time data

analysis and recording from a global peptide lists.

Results

To determine performance of th

of non-depleted human plasma

evaluation metrics was confide

matching, %CVs were used to

per method.

Data Analysis

Crystal spectral libraries were

identified and verified based o

years and contains 10,288 pep

and without modifications), rel

ion m/z values and average pr

perform real-time spectral mat

spectral library information res

Real-time identification was ba

mass errors, and cosine simila

distribution and spectral library

for all pSMART data and CS s

processed using two different

threshold of 0.7. Further scori

matches based on mass accur

was exported to the Pinpoint™

all technical replicates.

In addition to forward matching

subsequent data analysis. Th

decoy databases with the first

product ions. The two peptide

but precursor m/z value differe

extended the first by further sh

Decoy hits were scored using t

search.

FIGURE 1. Schematic repre

consisting of HR/AM MS spe

analysis

(red lines)

and loop

qualitative peptide confirmat

dictates MS acquisition cycl

precursor isolation range, a

cycle time. The real-time dat

showing the predicted retent

Library, precursor XIC, and t

precursor XIC trace.

Full Scan MS1 cycle

time

Narrow DIA cycle

Isolated/Filtered Precursor Mass Range

400

500

600

1200

…