AI-64310-CRFT

Thermo Scientific Poster Note

•

PN-64149-ASMS-EN-0614S

uisition scheme for performing

is. Evaluate data acquisition

ods.

S used for quantitative analysis

onfirmation. Utilize

time data analysis to create a

l replicates.

ore peptides identified and

ly fewer decoy matches

nd quantitative sample

MS-driven MS/MS

of peptides across the gradient

s which can then determine

ta independent acquisition

ity to archive and interrogate

ease the sampling and

ents, we utilize high

metric DIA windows as

used for quantitative and

ess is predicated on leveraging

mass accuracy to increase

quisition strategy enables

ause only one high quality

row precursor isolation using

gs have shown greater

on the same samples.

f human plasma collected

abilized tube (Becton

an plasma was prepared

ols following reduction and

as estimated to be 4 mg/µL,

ntil used. Before MS analysis,

ibration (PRTC) peptides

tration of 20 fmol on column.

hermo Scientific

EASY-

.2% formic acid in water and(B)

nto a 120 x 0.15 mm trapping

bs) and the analytical

packed with C18 Aq

ith a linear gradient from 5 to

mn regeneration.

as used for all experiments.

and peptide-based.

ime (pSMART) for data

25 Da precursor isolation

x ion fill times, 1e6 AGC

RT acquisition settings for MS

0) and DIA events were

r a precursor range of m/z

00-1200. Each narrow DIA

C settings, and 35,000

used to perform real-time data

Results

To determine performance of the pSMART acquisition strategy, experimental analysis

of non-depleted human plasma digest was compared to standard DIA data. The

evaluation metrics was confident matching as described above. In addition to spectral

matching, %CVs were used to determine reproducibility and quantitative capabilities

per method.

Data Analysis

Crystal spectral libraries were used to create a comprehensive list of plasma peptides

identified and verified based on DDA data acquired at BRIMS over the course of two

years and contains 10,288 peptides. The list of peptides contains the sequence (with

and without modifications), relative retention times, precursor charge states, product

ion m/z values and average product ion distribution. A custom script was used to

perform real-time spectral matching for both standard DIA and pSMART data to

spectral library information resulting in a final list of identified peptides per injection.

Real-time identification was based on retention time overlap, precursor/product ion

mass errors, and cosine similarity scoring between experimental product ion

distribution and spectral library information. Mass tolerance values were set to 10 ppm

for all pSMART data and CS scores of 0.6 or better. Standard DIA data was

processed using two different mass tolerance values, 10 and 20 ppm and a CS score

threshold of 0.7. Further scoring for standard DIA evaluated the consecutive spectral

matches based on mass accuracy and CS scores. The final list of identified peptides

was exported to the Pinpoint™ software for quantitation and variance analysis across

all technical replicates.

In addition to forward matching analysis, a decoy database was created and used for

subsequent data analysis. The same spectral library was used to create two different

decoy databases with the first decoy database created by switching precursors and

product ions. The two peptide entries used to switch must have similar retention times

but precursor m/z value differences in excess of 50 Da. The second decoy database

extended the first by further shuffling the relative abundance values per fragment ion.

Decoy hits were scored using the same acceptance criteria as that for the forward

search.

FIGURE 1. Schematic representation of pSMART data acquisition strategy

consisting of HR/AM MS spectral acquisition used for quantitative peptide

analysis

(red lines)

and looped narrow asymmetrical DIA acquisition for

qualitative peptide confirmation (black dashes). (1A) User defined loop count

dictates MS acquisition cycle time while precursor m/z range, individual DIA

precursor isolation range, and max ion fill times dictate total DIA acquisition

cycle time. The real-time data processing scheme is displayed in Fig. 1B

showing the predicted retention time window read in from the Crystal Spectral

Library, precursor XIC, and the single narrow DIA window acquired under the

precursor XIC trace.

Full Scan MS1 cycle

time

Retention Time (min)

Narrow DIA cycle time

Isolated/Filtered Precursor Mass Range

400

500

600

1200

…

Retention Time

Expected Retention Time

32.5

33.5

Normalized Intensity

Retention Time

100

(8.E+06)

100

Standard

pSMART

(7.E+05)

MS1

DIA

FIGURE 2. Comparative targeted p

pSMART methods for the plasma

ion XIC results from the standard

precursor ion XIC results used to

and quantitation. The dashed line

spectrum for the targeted peptide.

distribution analysis for DIA and p

distribution read in from Crystal.

in excess of 0.7 as compared to lib

DIA is 0.87 compared to 0.81 for p

relative product ion AUC values w

used to calculate the CS score.

TABLE 1. List of comparative pepti

strategies. Each column lists the f

acceptance criteria used for proce

pSMART data (MS and DIA) was 10

experimental data are described a

Peptide

IDStrategy

Standard DIA

20ppm

6 data point

across peak

Stan

4 da

acro

Library Hits

2159

Decoy library

#1 Hits

983

Decoy library

#2 Hits

515

Decoy Hit

Rate (#1)

46%

Decoy Hit

Rate (#2)

24%