PARAFFIN - EMBEDDED SAMPLES FOR SHOTGUN PROTEOMIC ANALYSIS OF FORMALIN - FIXED , C RITICAL COMPARISON OF SAMPLE PREPARATION STRATEGIES

(1)

C ^RITICAL ^COMPARISON ^OF ^SAMPLE PREPARATION STRATEGIES

FOR SHOTGUN PROTEOMIC ANALYSIS OF FORMALIN - ^FIXED ,

PARAFFIN - ^EMBEDDED ^SAMPLES

**Marcello Abbondio, Alessandro Tanca*, Salvatore Pisanu,** Sergio Uzzau, Daniela Pagnozzi, Maria Filippa Addis

Porto Conte Ricerche Srl, Tramariglio, Alghero (SS), Italy; tanca@portocontericerche.it*

1. I NTRODUCTION

The growing field of formalin-fixed paraffin-embedded (FFPE) tissue proteomics holds promise for improving translational research. Worldwide archival tissue banks hold a significant number and variety of tissue samples, as well as a wealth of retrospective information regarding diagnosis, prognosis, and response to therapy. This makes them an important resource for protein biomarker discovery and validation. Direct tissue trypsinization (DT) and protein extraction followed by in solution digestion (ISD) or filter-aided sample preparation (FASP) are the most common workflows for shotgun LC- MS/MS analysis of FFPE samples. However, there is currently no consensus on the optimal protocol, and no studies critically comparing the performance of the three different methods with FFPE specimens have been reported so far. Liver tissue was chosen as a model in consideration of its high proteome complexity in terms of expressed proteins and metabolic pathways.

4. C ^ONCLUSIONS

These results highlight that diverse sample preparation strategies provide qualitatively and quantitatively different proteomic information, and present typical biases that should be taken into account when planning a shotgun proteomic investigation dealing with FFPE samples. In view of the considerable portion of unique identifications provided by each method (particularly by DT and FASP), when a sufficient amount of tissue is available, a complementary, parallel use of different sample preparation strategies is suggested to increase proteome coverage, width and depth.

3.2. Q

^UALITATIVE ^AND QUANTITATIVE COMPARISON

A B

1 10 100 1000

1 10 100 1000

Log10NSAF ISD

Log₁₀NSAF FASP

1 10 100 1000

1 10 100 1000

Log10NSAF ISD

Log₁₀NSAF DT

1 10 100 1000

1 10 100 1000

Log10NSAF FASP

r=0.952 r=0.897 r=0.941

DT FASP

ISD

1274

50.0%

317

124

314 302

171 47

FASP 1

FASP 2

FASP 3

ISD 3

ISD 1

ISD 2 DT 1

DT 3

DT 2

1 10 100

Log10NSAF ISD

Log₁₀NSAF FASP

1 10 100

Log10NSAF ISD

1 10 100

Log10NSAF FASP

r=0.775 r=0.576 r=0.622

DT FASP

ISD

3595

26.6%

2746

406

3387 2329

745 204

FASP 1

FASP 2

FASP 3 ISD 3

ISD 1

ISD 2 DT 1

DT 2

DT 3

C D

E F

Top: Unsupervised hierarchical cluster analysis based on protein (A) and peptide (B) label-free quantitative data, respectively.

Middle: Venn diagrams illustrating distribution of all identified proteins (C) and peptides (D). Percentage of common proteins and peptides are indicated in yellow.

Bottom: Dot plots describing correlation of protein (E) and peptide (F) abundance between DT and FASP, DT and ISD, FASP and ISD. Pearson correlation coefficients are also reported.

3. R ÊSULTS ÂND D ÎSCUSSION

179

235

61 112

1126

58.0%

174 53

DT 1 DT 2

DT 3

1 10 100 1000

Log10NSAF DT 3

Log₁₀NSAF DT 2

1 10 100 1000

Log10NSAF DT 3

Log₁₀NSAFDT 1

1 10 100 1000

Log10NSAF DT 2

Log₁₀NSAF DT 1

r=0.910 r=0.957 r=0.931

136 128

163 179

1353

65.2%

54 63

FASP 1 FASP 2

FASP 3

1 10 100 1000

Log10NSAF FASP 2

Log₁₀NSAF FASP 1

1 10 100 1000

Log10NSAF FASP 3

1 10 100 1000

Log10NSAF FASP 3

r=0.995 r=0.974 r=0.978

ISD 1 ISD 2

ISD 3

139

1124

69.5%

43 28

139 75

68

1 10 100 1000

Log10NSAF ISD 2

Log₁₀NSAF ISD 1

1 10 100 1000

Log10NSAF ISD 3

1 10 100 1000

Log10NSAF ISD 3

r=0.994 r=0.987 r=0.988

A

1462

1371

488 1126

2895

32.6%

1221 311

DT 1 DT 2

DT 3

1 10 100

Log10NSAF DT 3

Log10NSAF DT 2

1 10 100

Log10NSAF DT 3

Log10NSAF DT 1

1 10 100

Log10NSAF DT 2

Log10NSAF DT 1

r=0.582 r=0.561 r=0.488

960 1003

1310 1825

4623

44.1%

330 422

FASP 1 FASP 2

FASP 3

1 10 100

Log10NSAF FASP 3

Log10NSAF FASP 2

1 10 100

Log10NSAF FASP 3

1 10 100

Log10NSAF FASP 2

r=0.920 r=0.705 r=0.728

ISD 1 ISD 2

ISD 3

691

2798

56.5%

214 113

519 268

347

1 10 100

Log10NSAF ISD 3

Log10NSAF ISD 2

1 10 100

Log10NSAF ISD 3

Log10NSAF ISD 1

1 10 100

Log10NSAF ISD 2

Log10NSAF ISD 1

r=0.899 r=0.833 r=0.822

B

3.1. R

EPRODUCIBILITY

• lower reproducibility

• good preservation of high-MW proteins

• much lower keratin contamination

• higher abundance of non tryptic peptides

• depletion of high-MW proteins

• enrichment in hydrophobic and membrane proteins

• higher identification yields

• higher reproducibility DT

FASP AND ISD FASP

ISD

Qualitative and quantitative reproducibility of DT, FASP and ISD.

A) Top: distribution of identified proteins among replicates. Percentage of common proteins are indicated in yellow.

Bottom: correlation of protein abundance between all replicates combinations for every method. Pearson correlation coefficients are also reported.

B) Same as Panel A but at peptide level.

3.5. N

^ON

-

^TRYPTIC ^AND FORMALDEHYDE

-

^MODIFIED ^PEPTIDES

DT FASP

ISD 75 78

129 226 525

37 37

DT FASP

ISD 10 25

76 117 270

12 8

A

B

DT

187 317

DT 8687

+3.6%

mod no mod

715 DT

7735 1139

trypsin DT no enzyme

+7.3% ¹⁸²² ⁸⁶⁵¹ ⁴¹⁶

FASP FASP

trypsin no enzyme

+3.8%

437

FASP FASP

10036 160

+1.5%

no mod mod

3734 278

1216

ISD

trypsin ISD no enzyme

+5.3%

4745 205

ISD ISD

+2.1% 106

mod no mod

A) Left: distribution of peptides identified with ‘trypsin’ and ‘no enzyme’ searches in DT, FASP and ISD samples. Right: distribution of non-tryptic peptides among all methods.

B) Left: distribution of peptides identified with standard search (‘no mod’) and search comprising formaldehyde-induced modifications (‘mod’) in DT, FASP and ISD samples. Right:

distribution of formaldehyde-modified peptides among all methods.

3.4. Q

UANTITATIVE PROTEIN DISTRIBUTION

:

PHYSICOCHEMICAL FEATURES

0 5 10 15 20 25 30 35 40 45 50

0-10 10-20 20-30 30-40 40-50 50-60 60-70 70-80 80-90 90-100 100-150 150-200 >200

% NSAF

MW (kDa)

*

** *

*

* * ** *

* * * ** *

** ** * ** * * * * ** *

*

0 5 10 15 20 25 30

<5 5-6 6-7 7-8 8-9 9-10 10-11 >11

% NSAF

pI

*

* *

*

* *

*

* ** *

*

0.0 0.1 0.2 0.3 0.4 0.5 0.6

GRAVY >0.5

% NSAF

*

0 1 2 3 4 5 6 7 8 9

TMD>0 TMD>1 TMD>2

% NSAF

*

**

* *

*

A B C D

0.0 0.2 0.4 0.6 0.8 1.0 1.2

GRAVY >0.5 GRAVY >0.5

proteins NSAF

%

DT FASP ISD

*

Quantitative protein distribution according to MW (A), pI (B), number of transmembrane domains (TMD, C) and hydrophobicity (GRAVY score, D). Mean and SD value of NSAF percentage for three independent experimental replicates are shown. NSAF values were expressed as percentage of all proteins.

Asterisks indicate statistical significance according to Student’s t-test (p value < 0.05): statistically significant difference versus DT, versus FASP, versus ISD and versus all other methods.

* * * *

0.0 0.5 1.0 1.5 2.0

0-10 10-20 20-30 30-40 40-50 50-60 60-70 70-80 80-90 90-100 100-150 150-200 >200

% NSAF

MW (kDa)

*

* **

*

* * ** *

* * * ** *

** ** *

* *

*

* *

*

0.0 0.5 1.0 1.5 2.0

0-10 10-20 20-30 30-40 40-50 50-60 60-70 70-80 80-90 90-100 100-150 150-200 >200

% NSAF

MW (kDa)

*

* **

*

* * ** *

* * * ** *

* * ** *

* *

*

* *

*

3.3. Q

UANTITATIVE PROTEIN DISTRIBUTION

: S

^UBCELLULAR LOCALIZATION

Mean and SD value of NSAF percentage for three independent experimental replicates are shown. NSAF values were expressed as percentage of the annotated proteins.

Asterisks indicate statistical significance according to Student’s t-test (p value < 0.05):

statistically significant difference versus DT versus FASP

versus ISD

versus all other methods

* *

0 500 1000 1500 2000 2500 3000 3500 4000 4500

Extracellular matrix Secreted Membrane Multi-pass membrane protein Single-pass membrane protein Peripheral membrane protein Lipid-anchor Cell membrane Cytoplasm Cytosol Cytoskeleton Nucleus Nucleus membrane Nucleus matrix Nucleolus Endoplasmic reticulum membrane Endoplasmic reticulum lumen Golgi apparatus Mitochondrion outer membrane Mitochondrion matrix Mitochondrion inner membrane Lysosome

NSAF

DT FASP ISD

*

* *

*

**

*

* **

*

**

* *

**

*

**

*

**

*

**

** *

***

* *

*

**

*

2. M ^ETHODS

0 1 2 3 4 5 6

Categoria 1 Categoria 2 Categoria 3 Categoria 4

D

^IRECT ^TISSUE TRYPSINIZATION

(DT)

Ammonium bicarbonate 50 mM

FASP

Microcon YM-30

I

^N ^SOLUTION ^DIGESTION

(ISD)

Detergent Removal Spin Columns

P^ROTEIN E^XTRACTION

SDS 2 %, DTT 200 mM, Tris–HCl (pH 8.8) 20 mM 99 °C for 60 min

DEPARAFFINIZATION

& R^EHYDRATION

T^RYPSIN DÎGESTION PÊPTIDE MÎXTURE LC-MS/MS

UltiMate 3000 RSLCnano LC system 485 min gradient

LTQ Orbitrap Velos - HCD

P^ROTEIN IDENTIFICATION Search engine: Sequest-HT

Peptide validation: Percolator

FDR < 1 % based on peptide q-value

D^ATA A^NALYSIS

Label free quantification via spectral counting Multivariate statistics using Perseus

C^OMPARISON

Reproducibility

Qualitative-quantitative Subcellular localization, pI, MW, GRAVY, TMD Formaldehyde-modified and non-tryptic peptides

H^UMAN ^LIVER ^TISSUE

3 INDEPENDENT REPLICATES PER METHOD

5 5-μ^M-^THICK ^SLICES ^PER ^REPLICATE

NSAF = Spc/L

∑ SpC/L

5. R

^EFERENCES

•Tanca A, Abbondio M, Pisanu S, Pagnozzi D, Uzzau S, Addis MF: Critical comparison of sample preparation strategies for shotgun proteomic analysis of formalin-fixed, paraffin-embedded samples: insights from liver tissue. Clin Proteomics 2014, 11(1):28.

•Tanca A, Pagnozzi D, Addis MF: Setting proteins free: Progresses and achievements in proteomics of formalin-fixed, paraffin- embedded tissues. Proteomics Clin Appl 2012, 6:7–21.

•Zybailov B, Mosley AL, Sardiu ME, Coleman MK, Florens L, Washburn MP: Statistical analysis of membrane proteome expression changes in Saccharomyces cerevisiae. J Proteome Res 2006, 5:2339–2347.

•Ostasiewicz P, Zielinska DF, Mann M, Wisniewski JR: Proteome, phosphoproteome, and N-glycoproteome are quantitatively preserved in formalin-fixed paraffin-embedded tissue and analyzable by high-resolution mass spectrometry. J Proteome Res 2010, 9:3688–3700.

•Alkhas A, Hood BL, Oliver K, Teng PN, Oliver J, Mitchell D, Hamilton CA, Maxwell GL, Conrads TP: Standardization of a sample preparation and analytical workflow for proteomics of archival endometrial cancer tissue. J Proteome Res 2011, 10:5264–5271.

•Gamez-Pozo A, Ferrer NI, Ciruelos E, Lopez-Vacas R, Martinez FG, Espinosa E, Vara JA: Shotgun proteomics of archival triple- negative breast cancer samples. Proteomics Clin Appl 2013, 7:283–291.

PARAFFIN - EMBEDDED SAMPLES FOR SHOTGUN PROTEOMIC ANALYSIS OF FORMALIN - FIXED , C RITICAL COMPARISON OF SAMPLE PREPARATION STRATEGIES

C RITICAL COMPARISON OF SAMPLE PREPARATION STRATEGIES

FOR SHOTGUN PROTEOMIC ANALYSIS OF FORMALIN - FIXED ,