Issuu on Google+

Targeted Protein Quantitation Triple Quadrupole Mass Spectrometry


Targeted Protein Quantitation Multiple Reaction Monitoring (MRM)

Application of Multiple Reaction Monitoring mode on a triple quadrupole mass spectrometer to a Liquid Tissue® lysate provides a very sensitive and highly specific assay protocol for quantitation of target proteins in formalin fixed tissue. The following two approaches are used: A Relative Quantitation • Compare quantitative amounts of specific, defined peptides of target proteins across multiple samples based on peak height variability. This can be accomplished either without labels or by labeling test sub-sets, e.g. with iTRAQ, mTRAQ B Absolute Quantitation • Determine absolute amount of targeted native peptides in a single Liquid Tissue® sample by comparison to spiked synthetic, labeled peptides of the same amino acid sequence that serve as internal standards


Relative Quantitation

A

RT: 7.97 - 16.21

Relative Abundance Abundance

Liquid Tissue速

RT: 11.91 MA: 6699

100 90 80 70 60 50 40 30 20 10 0

Sample 1 RT: 11.87

RT: 7.97 - 16.21

Relative Relative Abundance

Liquid Tissue速

RT: 11.91 MA: 6699

100 90 80 70 60 50 40 30 20 10 0

Sample 2 RT: 11.87

RT: 7.97 - 16.21

Relative Abundance

Liquid Tissue速

B

RT: 11.91 MA: 6699

100 90 80 70 60 50 40 30 20 10 0

Sample 3 RT: 11.87

Absolute Quantitation RT: 11.91 MA: 6699

100 90 70 60 50 40 30

2.50

Analyte Area/IS Area

Liquid Tissue速

Relative Abundance

80

20 10 0

RT: 11.87 MA: 284491

100 90 80 70 60 50 40 30 20

2.00

y = 0.189x - 0.0224 R 2 = 0.9997

1.50

1.00

0.50

0.00 0.00

2.00

4.00

6.00

8.00

fmols on column

10 0

8

9

10

11 12 13 14 Time (min)

15

16

10.00

12.00


Relative Quantitation MRM Validation of Candidate Biomarkers Experiment: A total of 9 stage IA (early stage) and 9 stage IIIA (metastatic) lung cancer tissue samples were microdissected and Liquid Tissue速 samples prepared and analyzed by LC-MS/MS, A Spectral count data was used to determine global differential protein expression linked to these specific stages of lung cancer. MRM analysis based on peak height variability of 14 proteins identified in B was performed across additional multiple lung cancer tissues of the same stages to specifically identify differentially expressing diagnostic/prognostic biomarkers of metastatic lung cancer. Results: More than 14,000 peptides were analyzed across 18 samples and 14 candidate proteins were chosen for relative quantitative comparisons. Peak height analysis of 14 individual peptides, one specific to each protein, suggests that NapsinA is prognostic for long term survival of stage IIIA lung cancer. As shown in C, peak height results for each sample were placed on a log scale and patients identified by high expression of NapsinA (blue) survived at least 22 months post surgery while those with reduced expression (red) succumbed to their cancer within 22 months. Expression of NapsinA was also determined in cancer cells microdissected from surrounding cancer-bearing lymph nodes and results are consistent with results from the primary tumors. Further analysis as shown in the Kaplan-Meier curve indicates a strong correlation between higher NapsinA expression and long term cancer survival for as long as 100 months D. Conclusions: 1. Global profiling of Liquid Tissue速 lysates provided for candidate biomarkers of metastatic lung cancer that were further analyzed for relative quantitative measure across many samples using MRM methodology. 2.

NapsinA was further characterized by MRM quantitative methodology as prognostic for long-term survival (100 months) of lung cancer.


C

1.0E+06

1.0E+05

1.0E+04

DEATH

Log 10 NAPSA_(506.8/553.3)

22 Month Survival

SURVIVAL

A

1.0E+03

1.0E+02

IA primary

IIIA primary

IIIA-lymph node

100

Kaplan-Meier curve 100 Month Survival

D 80

Survival Rate, %Rate % Survival

B

NAPSA (+) 60

40

NAPSA (-) 20

0 0

20

40

60

Month Months

80

100


Absolute Quantitation MRM Quantitation of Her2 in Breast Cancer Experiment: An MRM-based absolute quantitation assay using a spiked internal peptide standard was developed for the Her2 protein. Cancerous epithelium from 10 breast cancer showing a range of Her2 expression by IHC was microdissection and Liquid Tissue速 lysates prepared for MRM analysis. In addition to IHC and MRM, FISH was performed on each sample. MRM results are shown as fmol/袖g of protein analyzed, FISH results are indicated by a ratio of gene amplification, and IHC results as a score of 0-3+. MRM, IHC and FISH results were directly compared across all samples. Normalized MRM and FISH scores were determined by dividing each individual score by the average of all scores across all samples. Replicate analyses were performed on 3 serial sections in duplicate to assess reproducibility and compared to surrounding normal epithelium. Results: As shown in A, MRM analysis indicates strong correlation with existing IHC and FISH results across the entire tissue cohort, but with greater dynamic scoring range from none detected in samples 13 to 4.5 fmol/ug protein. Normalization of the MRM and FISH scores as shown in B was developed to directly compare disparate forms of data and results indicate tight correlation again, with absolute correlation for samples 7 & 8, both scored as IHC 2+ and where the FISH score for sample 7 is 1.6 while the sample 8 score is 2.1. MRM analysis of three replicates of a 3+ sample as shown in C indicates reproducibility across multiple serial sections of the same tissue sample with no detection of Her2 expression in surrounding normal epithelium. Conclusions: 1. Absolute quantitation of the Her2 protein in FFPE tissue utilizing Liquid Tissue速 reagents indicates strong correlation of protein expression to IHC in a breast cancer tissue cohort. 2.

Results demonstrate strong correlation to FISH data of the same samples and normalization of the data indicate exact correlation to clinically relevant data in the IHC 2+ samples.

3.

The assay is reproducible across multiple sections cut from the same block as well as reproducible within the same Liquid Tissue速 prep.


A

C Absolute Quantitation - MRM vs. FISH vs. IHC

2

IHC score

0

MRM score (fmol/ug protein)

4.5

Reproducibility of Single Sample

14 12

MRM Score FISH Score

10 8 6

10.00

IHC = 3+ FISH = 12.5

FISH Score

2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 Samples 1

H&E

4

8.00

Her2 fmolprotein) / ug Her2 peptide ((fmol/ug

2 0 3

4

5

6

7

1+

8

9

2+

10 3+

Normalized Scores - MRM vs. FISH

B

Normalized MRM Score Normalized FISH Score

IHC Score

2 0

3

4

5 1+

6

7

8 2+

NCI/EPI patent application filed Feb. 2007

9

10 3+

Normalization Score

5.5 5.5 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0

6.4

Samples 1

Cancer

IHC

6.00

4.00

Normal H&E 2.00

Not Detected

0.00 3+

Section 96-63-C 1

3+

Section 96-63-C 2

3+

NA

Section Normal 96-63-C 96-63-B 3

Sl i de #1

Sl i de #2

Sl i de #3

80605E S-1

80605E S-2

80605E S-3

80912E S-4


Collaborative Assay Development Project Absolute Quantitation of Novel Drug Target Experiment: A labeled peptide Liquid Tissue速 MRM assay was developed for a proprietary drug target protein for which an immunohistochemistry assay in formalin fixed tissue was not able to be developed. Liquid Tissue速 preparations of the purified form of the protein and a cell line expression the target protein were analyzed to determine specific tryptic peptides that could be efficiently detected by mass spectrometry. Two peptides were chosen to develop an absolute quantitative assay for this drug target and the assay was applied to Liquid Tissue速 preparations from the formalin fixed cell line not expressing the drug target, the formalin fixed cell line transfected with and expressing the drug target, and a formalin fixed xenograph tumor from the expressing cell line. Results: The assay detects neither peptide in the negative control cell line while the transfected cell line indicates quantitation of 995 amol/ug for peptide 1 and 1076amol/ug for peptide 2. Analysis of the xenograph lysate demonstrates 1162amol/ug for peptide 1 and 1130 1076amol/ug for peptide 2. For those samples analyzed in triplicate, CVs of <6% was observed for each peptide. Conclusions: 1. A 2-peptide Liquid Tissue速 MRM assay that provides for absolute quantitation of a novel drug target was developed in 6 weeks and successfully demonstrated in formalin fixed test samples. 2.

Quantitative results are identical between the transfected cell line expressing the drug target and a xenograph tumor derived from this cell line with little variation between replicates. In addition, the negative control cell line showed no detected expression.


Peptide 1

Peptide 2

Not Detected

Not Detected

Transfected Cell Line formalin fixed

995 amol/ug (+/- 6.7amol)

1076 amol/ug (+/- 59amol)

Xenograph Tumor formalin fixed

1162 amol/ug (+/- 67amol)

1130 amol/ug

Cell Line formalin fixed

• Client unable to develop IHC assay in FFPE tissue for drug target of interest • Partnered with EPI to develop a quantitative protein assay using MRM platform • Successful 2-peptide assay demonstrated in 6 weeks


www.expressionpathology.com


Protein Quantitation with EPI Platform