CRITICAL 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. INTRODUCTION 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. CONCLUSIONS 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. QUALITATIVE AND QUANTITATIVE COMPARISON

A B

1

10

100

1000

1 10 100 1000

Log 1

0N

SAF

ISD

Log10 NSAF FASP

1

10

100

1000

1 10 100 1000

Log 1

0N

SAF

ISD

Log10 NSAF DT

1

10

100

1000

1 10 100 1000Log 1

0N

SAF

FASP

Log10 NSAF DT

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

1 10 100

Log 1

0N

SAF

ISD

Log10 NSAF FASP

1

10

100

1 10 100

Log 1

0N

SAF

ISD

Log10 NSAF DT

1

10

100

1 10 100Log 1

0N

SAF

FASP

Log10 NSAF DT

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. RESULTS AND DISCUSSION

179

235

61

112

1126

58.0% 174 53

DT 1 DT 2

DT 3

1

10

100

1000

1 10 100 1000

Log 1

0N

SAF

DT

3

Log10 NSAF DT 2

1

10

100

1000

1 10 100 1000

Log 1

0N

SAF

DT

3

Log10 NSAF DT 1

1

10

100

1000

1 10 100 1000

Log 1

0N

SAF

DT

2

Log10 NSAF 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

1 10 100 1000Log 1

0N

SAF

FASP

2

Log10 NSAF FASP 1

1

10

100

1000

1 10 100 1000Log 1

0N

SAF

FASP

3

Log10 NSAF FASP 1

1

10

100

1000

1 10 100 1000Log 1

0N

SAF

FASP

3

Log10 NSAF FASP 2

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

1 10 100 1000Log 1

0N

SAF

ISD

2

Log10 NSAF ISD 1

1

10

100

1000

1 10 100 1000Log 1

0N

SAF

ISD

3

Log10 NSAF ISD 1

1

10

100

1000

1 10 100 1000Log 1

0N

SAF

ISD

3

Log10 NSAF ISD 2

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

1 10 100

Log 1

0N

SAF

DT

3

Log10 NSAF DT 2

1

10

100

1 10 100

Log 1

0N

SAF

DT

3

Log10 NSAF DT 1

1

10

100

1 10 100

Log 1

0N

SAF

DT

2

Log10 NSAF 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

1 10 100Log 1

0N

SAF

FASP

3

Log10 NSAF FASP 2

1

10

100

1 10 100Log 1

0N

SAF

FASP

3

Log10 NSAF FASP 1

1

10

100

1 10 100Log 1

0N

SAF

FASP

2

Log10 NSAF FASP 1

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

1 10 100Log 1

0N

SAF

ISD

3

Log10 NSAF ISD 2

1

10

100

1 10 100Log 1

0N

SAF

ISD

3

Log10 NSAF ISD 1

1

10

100

1 10 100Log 1

0N

SAF

ISD

2

Log10 NSAF ISD 1

r=0.899 r=0.833 r=0.822

B

3.1. REPRODUCIBILITY

• 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. NON-TRYPTIC AND FORMALDEHYDE-MODIFIED PEPTIDES DT FASP

ISD

75

78

129

226 525

37 37

DT FASP

ISD

10

25

76

117 270

8 12

A

B DT

187 317

DT

8687 +3.6%

mod no mod

715

DT

7735 1139

DT trypsin no enzyme

+7.3% 8651 416 1822

FASP FASP trypsin no enzyme

+3.8%

437

FASP FASP

10036 160 +1.5%

no mod mod

3734 278 1216

ISD ISD trypsin no enzyme

+5.3%

4745 205

ISD ISD

106 +2.1%

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. QUANTITATIVE 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

% N

SAF

MW (kDa)

*

** *

*

*

* * ** *

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

*

*

0

5

10

15

20

25

30

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

% N

SAF

pI

*

*

**

*

*

**

*

*

** *

* **

*

0.0

0.1

0.2

0.3

0.4

0.5

0.6

GRAVY >0.5

% N

SAF

*

*

*

0

1

2

3

4

5

6

7

8

9

TMD>0 TMD>1 TMD>2

% N

SAF

*

**

**

* **

*

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

% N

SAF

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

% N

SAF

MW (kDa)

*

* * *

*

*

* * * * ** * * * **

** * **

* *

*

*

*

**

* *

*

*

3.3. QUANTITATIVE PROTEIN DISTRIBUTION: SUBCELLULAR 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. METHODS

0

1

2

3

4

5

6

Categoria 1 Categoria 2 Categoria 3 Categoria 4

DIRECT TISSUE TRYPSINIZATION (DT) Ammonium bicarbonate 50 mM

FASP Microcon YM-30

IN SOLUTION DIGESTION (ISD) Detergent Removal Spin Columns

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

DEPARAFFINIZATION & REHYDRATION

TRYPSIN DIGESTION PEPTIDE MIXTURE LC-MS/MS UltiMate 3000 RSLCnano LC system 485 min gradient

LTQ Orbitrap Velos - HCD

PROTEIN IDENTIFICATION Search engine: Sequest-HT

Peptide validation: Percolator

FDR < 1 % based on peptide q-value

DATA ANALYSIS Label free quantification via spectral counting

Multivariate statistics using Perseus

COMPARISON Reproducibility

Qualitative-quantitative

Subcellular localization, pI, MW, GRAVY, TMD

Formaldehyde-modified and non-tryptic peptides

HUMAN LIVER TISSUE 3 INDEPENDENT REPLICATES PER METHOD 5 5-μM-THICK SLICES PER REPLICATE

NSAF = Spc/L

∑ SpC/L

5. REFERENCES •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.