AQUA peptides, QCAT concatemers, and

full-length isotope-labeled proteins:

Which choice for protein absolute

quantification ?

Virginie Brun

CEA/INSERM U880 Etude de la Dynamique des Protéomes, Grenoble

Protein absolute quantification

Definition: Exact determination of a protein concentration in a sample

Targeted approach

Applications:

Qualification of biomarkers

Medical diagnosis

Quality control

Analytical performances required:

Accuracy

Precision reproducibility, repeatability

Linearity in a (large) dynamic range

Sensitivity false negatives

Specificity false positives

Principle of protein absolute quantification

using isotopic dilution and MS

1. Selection of marker peptides (LC-MS/MS and LC-MS)

Selection parameters:

Sequence uniqueness specificity

No M, C, W residues

Retention time

Ionization capacity

No miscleavages

2. Standardization

= Addition into the samples of a defined quantity of

quantification standards. These standards correspond to (or

generate)the same marker peptides as the target proteins.

However, one/several AA is/are isotopically labeled.

Same chemicophysical properties but mass increasem/z

im/z

Peptide

marqueur

natif

Peptide

marqueur

alourdi

m/z

im/z

m/z

im/z

Peptide

marqueur

natif

Peptide

marqueur

alourdi

Marker peptide (light)

Standard peptide (heavy)

Absolute quantification tools

AQUA peptides Stemmann et al., Cell, 2001; Gerber et al., PNAS, 2003

QCAT/polySIS concatemers Beynon et al., Nat. Methods, 2005

Full-length isotope-labeled proteins (PSAQ) EDyP Patent

AQUA peptides

Isotope-labeled peptides chemically synthetized

2 suppliers: Sigma + Thermo

500 $ for 5 nanomoles

Recommendations:

Advantage: possible phosphorylation(s)

Use: Add before digestion (in solution) or before LC-MS analysis

Supplier:

• Defined labeling: R, I, L, K, F, P and V

• Length 15 AA

• No chemically M, C, W, N-ter.Q

(chemical reactivity)

Our team:

• High MS detectability at low

concentrations

• Avoid peptides generating multiple ion

forms

• Avoid C-ter labeling

AQUA peptides

Selected application

Absolute quantification of cyclin-dependent kinases multisite phosphorylation

From Mayya et al., MCP 2006

Jurkat cells, IP of Cdk1 and Cdk2

IGEGTYGVVYK detected by SRM

4 phosphorylated/unphosphorylated versions

Phosphorylation inhibitory effect

M PhaseG1, S, G2 Phase

AQUA peptides

Pitfalls

Absolute quantification of C-reactive protein in the serum of patients

with rheumatoid arthritis

From Kuhn et al., Proteomics, 2004

Processed healthy serum sample containing exogenous CRP (121 µg/mL)

CRP concentration by MRM

(µg/mL)

Recovery

(%)

Marker peptide 1 83.3 66

Marker peptide 2 35.4 28

Marker peptide 3 0.23 0.2

Marker peptide 4 30.4 24

• Serum depletion

• Size exclusion chromatography

• DTT/iodoacetamide

• Size fractionation

• SDS-PAGE

• trypsin digestion

• MRM

• NO ASSESSMENT of analyte losses during the prefractionation steps

• NO ASSESSMENT of tryptic digestion yield

• Solubility/conservation of AQUA peptides

QCAT/polySIS concatemers

QCAT = concatemer of isotope-labeled marker peptides

Design of QCAT protein:

Synthesis of QCAT gene:

Retro-translation

Synthesis of 5’ phosphorylated overlapping oligonucleotides

Ligase Chain Reaction + PCR artificial gene

QCAT gene cloning into an expression plasmid

Expression and labeling:

In vivo or in vitro

ARG and LYS labeling preferred

Purification / quantification

Use: Add before tryptic digestion or eventually before SDS-PAGE

From Beynon et al., Nat. Methods, 2005

QCAT/polySIS concatemers

Selected application

Absolute multiplexed quantitative analysis of protein expression

during muscle development using QconCAT

From Rivers et al., MCP, 2007

Pro

tein

(nm

ol/g tis

sue)

• Quantification of chicken muscle proteins during growth

• Soluble fraction of pectoralis muscle

• Preliminary optimization of proteolysis conditions

• Addition of QCAT= concatemer of 20 tryptic peptides from 20 chicken muscle proteins

• Trypsin digestion

• LC-MS analysis

QCAT/polySIS concatemers

Pitfalls

Efficiency of labeling

labeling + metabolic scramble in vivo

Use: predigested or codigested ?

Differential sensitivity of to proteolysis

y = 0.5429x

R2 = 0.999

0

50

100

150

200

250

300

350

400

450

0 100 200 300 400 500 600

Injected SEA (pg)

Esti

mate

d S

EA

(p

g)

QCAT Analytes

• NO ASSESSMENT of analyte losses during the

prefractionation steps

• RISK : Analyte underevaluation in the case of

incomplete proteolysis

From Rivers et al., MCP, 2007

From Brun et al., MCP, submitted

Full-length isotope-labeled proteins (PSAQ)

PSAQ = Protein Standard Absolute Quantification

Cloning of genes into expression plasmids (with

cleavable tags)

Expression and labeling:

In vivo or in vitro

ARG and LYS labeling preferred

Purification, tag cleavage and quantification

Use: Add directly in the sample to be analyzed

PSAQ

Selected application

y = 0.2117x

R2 = 0.9805

y = 0.5795x

R2 = 0.9952

y = 1.0458x

R2 = 0.9924

0

20

40

60

80

100

120

0 20 40 60 80 100

Added SEA (ng/ml urine)

Es

tim

ate

d S

EA

(n

g/m

l u

rin

e)

y = 0.0347x

R2 = 0.758

y = 0.1885x

R2 = 0.9826

y = 1.0834x

R2 = 0.99

0

50

100

150

200

250

300

0 50 100 150 200 250

Added TSST-1 (ng/ml urine)

Es

tim

ate

d T

SS

T-1

(n

g/m

l u

rin

e)

Urine

sample

Protein capture

on Strataclean

resin

SDS-PAGE Trypsin

digestion

nanoLC-MS

analysis

A

B C

PSAQ standard

QCAT standard

AQUA standard

PSAQ standard

QCAT standard

AQUA standard

PSAQ

standards

QCAT

standard

AQUA

peptides

y = 0.2117x

R2 = 0.9805

y = 0.5795x

R2 = 0.9952

y = 1.0458x

R2 = 0.9924

0

20

40

60

80

100

120

0 20 40 60 80 100

Added SEA (ng/ml urine)

Es

tim

ate

d S

EA

(n

g/m

l u

rin

e)

y = 0.0347x

R2 = 0.758

y = 0.1885x

R2 = 0.9826

y = 1.0834x

R2 = 0.99

0

50

100

150

200

250

300

0 50 100 150 200 250

Added TSST-1 (ng/ml urine)

Es

tim

ate

d T

SS

T-1

(n

g/m

l u

rin

e)

Urine

sample

Protein capture

on Strataclean

resin

SDS-PAGE Trypsin

digestion

nanoLC-MS

analysis

Protein capture

on Strataclean

resin

Protein capture

on Strataclean

resin

SDS-PAGE Trypsin

digestion

nanoLC-MS

analysis

nanoLC-MS

analysis

A

B C

PSAQ standard

QCAT standard

AQUA standard

PSAQ standard

QCAT standard

AQUA standard

PSAQ

standards

QCAT

standard

AQUA

peptides

Staphylococcal toxins

SEA and TSST-1

From Brun et al., MCP, submitted

Absolute quantification of Staphylococcus aureus toxins in urine

PSAQ

Pitfalls

Labeling efficiency

(labeling + metabolic scramble in vivo)

Post-translational modifications

Differential oxydation state/ folding between analyte and standard

Conclusion

AQUA peptides QCAT/polySIS concatemers

Full-length isotope-labeled proteins (PSAQ)

Accuracy - to +++ - to +++ +++

Precision +++ +++ +++

Execution Easy and short Difficult and long

or Entelechon GmbH

Easy and short/long

Compatibility with prefractionation

NO NO YES

Discrimination of variants/isoforms

+/- +/- ++

Study of protein complexes (stoichiometry)

+/- +++ ++

Post-translational modifications

+++ (phosphorylation)

NO NO

Cost Very high High Moderate to High

ALWAYS ASSESS

the completeness of proteolysis

PREFERRED APPLICATIONS:

AQUA peptides peptidomics and phosphorylation studies

QCAT concatemers protein complexes (stoichiometry)

PSAQ standards accurate quantification in prefractionated samples

Summary