Linear Peptide Response Non-Linear Peptide Response

Peptide Quality VennDiagram

100, 50, 25 fmol Isotope Dot Product > 0.95

Label-Free Peptide Dilution Experiment Distribution of Log-Ratios

Peptides were filtered if any IsotopeDotProduct > 0.5 (875 peptides remained, 138 filtered)

Rectangle Shaded Area is mean +/- one Standard Deviation

Fold Change

(relative to 100 fmol) Peptides were filtered if any IsotopeDotProduct > 0.5

(875 peptides remained, 138 filtered)

Dilution Ratios Peptides were filtered if any IsotopeDotProduct > 0.5 (875 peptides remained, 138 filtered)

Diagonal is histogram of log2peak Area

Off-diagonal is scatter plot between two log2 peak areas

ABRF-sPRG2013 Development and Characterization of a Proteomics Normalization Standard Consisting of 1,000 Stable Isotope Labeled Peptides

Christopher Colangelo1; Craig Dufresne2; Alexander Ivanov3; Antonius Koller4; Brett Phinney5; Kristie Rose6; Paul Rudnick7; Brian Searle8; Scott Shaffer9 1Yale University, New Haven , CT; 2Thermo Fisher Scientific, West Palm Beach, FL; 3Northeastern University, Boston, MA; 4Stony Brook University, Stony Brook, NY; 5University of California, Davis, CA; 6Vanderbilt University, Nashville, TN; 7National

Institute of Standards and Technology, Gaithersburg, MD; 8Proteome Software, Portland, OR; 9University of Massachusetts Medical School, Worcester, MA

A BR F

Participate in the sPRG2013 Study and have fun with quantitation and refining your analytical approaches!!! Visit www.abrf.org/sprg

Introduction Proteomics technologies are an integral part of biological and

clinical research. Significant contributions by the proteomics field

are driven by the diverse and advanced analytical approaches

employed to comprehensively characterize proteomes, including

quantitative analysis of proteome variations, modifications, and

interactions. The ABRF Proteomics Standards Research

Group (sPRG) functions to design and develop performance

standards and resources for mass spectrometry-based

proteomics applications. The sPRG is currently conducting

a study focused on generation of a standard for interassay,

interspecies, and interlaboratory normalization in label-free as

well as in quantitative stable-isotope label-based analyses. The

standard has been formulated as two mixtures: 1,000 stable

isotope 13C/15N-labeled synthetic tryptic peptides alone, and

peptides mixed with a tryptic digest from HEK 293 cell lysate.

Methods Sequences of synthetic peptides were derived from

approximately 552 proteins, conserved across proteomes of

commonly analyzed species: Homo sapiens, Mus musculus and

Rattus norvegicus. Peptides represent a wide range

of hydrophobicities and isoelectric points typical of complex

proteomics samples. The 1,000 isotope-labeled peptides were

spot-synthesized by jpt.com. Individual peptides were

reconstituted, combined and desalted by solid-phase extraction.

HEK cell lysates were prepared by RIPA lysis and sonication, and

proteins were methanol-precipitated, resolubilized, and digested

with Lys-C and trypsin. The HEK proteolytic peptide mixture was

desalted by solid-phase extraction. For the combined

formulation, 1 pmol of synthetic peptide was added to 5 μg of the

HEK peptide mixture. The synthetic peptide mixture and the

combined synthetic and HEK-derived peptide mixture

were characterized individually by sPRG members representing

multiple LC-MS/MS instrument platforms.

Conclusion The Proteomics Standards Research Group reports the progress

for the first year in development of an innovative proteomics

normalization standard, designed to represent proteins of various

concentrations and spanning three orders of magnitude. 1,000

isotope labeled peptides were synthesized and analyzed in the

laboratories of sPRG members with various instruments and

fragmentation methods. Greater than 99% of the peptides were

identified during the validation runs. In peptide dilution

experiments, the majority of peptides behaved as expected with a

linear response, however, a few peptides had a non-linear

response. The peptides were spiked into a HEK digest and

showed minimal variation in both retention time and peak area.

The sPRG is hopeful that the designed formulation will become a

valuable resource in various mass spectrometry-based proteomic

applications, including quantitative and differential protein profiling,

as well as general benchmarking (e.g. chromatographic retention

time) for LC-MS performance.

L S

L S M Y G V D L

T H S

K*

P F R*

K*= Lys (U-13C6;U-15N2), mass difference: + 8 Dalton

R*= Arg (U-13C6;U-15N4), mass difference: + 10 Dalton

ABRF sPRG Peptides

isotopically labeled, proteotypic peptides

that terminate with C-terminal heavy Arg/Lys

Instrument Platforms Used for Validation

Protein Identification of

ABRF Peptide Standards

Instrument LC IDA/SWATH Fragmentation

ABSciex 5600TT Waters NanoAcquity IDA QTOF

ABSciex 5600TT Waters NanoAcquity SWATH QTOF

Thermo Orbitrap-Elite Waters NanoAcquity IDA HCD

Thermo Orbitrap-Elite Waters NanoAcquity IDA CID(IT)

Thermo Orbitrap-XL Eksigent nano 2DLC IDA CID(IT)

Thermo Q-Exactive Waters NanoAcquity IDA HCD

0

0.2

0.4

0.6

0.8

1

1.2

0 200 400 600 800x f

mo

l p

ep

tid

e a

rea

/10

0fm

ol

pe

pti

de

are

a

peptide

Individual Peptide Peak Area (relative to 100 fmol)

6fmol

12fmol

25fmol

50fmol

100fmol

Characterization of Peptide Standards

0

10

20

30

40

50

60

70

-2.7

-2.4

-2.1

-1.8

-1.5

-1.2

-0.9

-0.6

-0.3

0 0.3

0.6

0.9

1.2

1.5

1.8

2.1

# o

f p

ep

tid

es

Gravy scores

0

50

100

150

200

250

# o

f p

ep

tid

es

% hydrophobicity (Bull Breese Index)

0

50

100

150

200

6 8 10 12 14 16 18 20

# o

f p

ep

tid

es

peptide length

0

100

200

300

400

500

1 2 3

# o

f p

rote

ins

# of peptides

0

20

40

60

80

100

700

-750

850

-900

100

0-1

05

0

115

0-1

20

0

130

0-1

35

0

145

0-1

50

0

160

0-1

65

0

175

0-1

80

0

190

0-1

95

0

205

0-2

10

0

220

0-2

25

0

# o

f p

ep

tid

es

Molecular weight (Da)

- Linear Fit

- isotope dot product > 0.95

- isotope dot product > 0.8

- isotope dot product > 0.5

Acknowledgements The ABRF Proteomics Standards Research Group (sPRG) would

like to thank JPT Peptide Technologies GmbH for providing greatly

discounted synthetic peptides. We also thank Thermo Scientific

and Proteome Software for their generous technical support.

Data Processing and Analysis 50 Data Dependent (IDA) and 12 Data Independent (SWATH) LC-MS runs were performed using 4

types on instruments at 5 laboratories. MASCOT mgf files were generated from all 50 IDA files

using MSConvert (Proteomewizard) on ThermoScientific *.raw files and AB Sciex MS Data

Converter 1.3 *.wiff files. Protein Identification was performed using MASCOT 2.4 and a

concatenated ABRF peptide sequence and the human Swiss-Prot database. MASCOT dat files

were converted to XML and uploaded to Yale Protein Expression Database. Label-Free Precursor

Ion Analysis was performed using Skyline 1.4. Briefly, MASCOT dat files were imported to

generate a spectral library. Raw datafiles were integrated and the peak area results were exported

to csv file which were imported into Excel and R 2.14.2 for downstream biostatistical analysis.

ABRF Peptides

Both

HEK

Label-Free Peptides + HEK

Distribution of Peptide Ion Intensities Isotope dot product > 0.95

R² = 0.9838

0

25

50

75

100

125

150

0 25 50 75 100 125 150

AB

RF

Pep

tid

es +

HE

K

ABRF Peptides

ABRF Peptides

Both

HEK

Estimated Abundance (Middle) Protein: MATR3_HUMAN

Peptide: TEEGPTLSYGR (2+)

Estimated HEK amount – 125 fmol

Estimated Abundance (High) Protein: NPM_HUMAN

Peptide: VDNDENEHQLSLR (2+, 3+)

Estimated HEK amount – 1500 fmol

Protein ID of HEK All seventeen HEK LC-MS IDA runs

were combined and searched with

MASCOT against the human

Swissprot database. The search

results were filtered to 1.0% FDR on

the peptide spectrum match level,

resulting in 4,171 distinct proteins

from 126,173 distinct peptide

identifications greater than identity.

0

200

400

600

800

1000

1200

0

10

20

30

40

50

60

70

80

90

100

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Pep

tid

es >

Id

en

tity

(1%

FD

R)

% o

f A

BR

F P

ep

tid

es I

den

tifi

ed

Sample

% Coverage Peptide score > Identity score

Retention Times are Reproducible

Between Samples

ABRF Peptides

500fmol

HEK

1 ug

ABRF Peptides + HEK

500fmol + 1 ug