Label-free differential analysis of proteins in lipid rafts using LC-MS and the MSIGHT software tool

Manfredo Quadroni1, Frederique Baruthio2, Daniel Walther3, Ron Appel3,Curzio Ruegg2 and Agnese Mariotti2

1,Protein Analysis Facility, University of Lausanne, Epalinges, Switzerland

2, Multidisciplinary Oncology Center, Swiss Institute for Experimental Cancer research (ISREC), Epalinges, Switzerland

3, Swiss Institute of Bioinformatics, Proteome Informatics Group, Geneva, Switzerland

www.unil.ch/paf

1Automated LC-MS/MS after proteolysis is one of the methods of choice for the comprehensive analysis of complex protein mixtures. Though powerful, this technique favors the identification of highly abundant proteins, while minor components of interest can be neglected. Also, automated data-dependent choice of precursors has a limited reproducibility in LC conditions.

There is therefore a need for CID-independent data analysis strategies for performing quantification and comparisons of complex samples. Isotope labelling schemes have greatly increased the scope of the classical LC-MS/MS workflows by allowing for reliable quantification. However isotope labellingstrategies are not always easily applicable and do not fully solve the problem of insufficient sampling of low-abundance components.

Here we explore the use of MSIGHT (3), a freeware software tool for the visualization, alignment and comparison of LC-MS datasets to perform detailed differential analysis and quantification of unlabelled protein components after digestion and LC-MS/MS. We have applied the strategy to compare the protein composition of lipid rafts isolated from three melanoma cell lines with different metastatic phenotypes .

Abstract

Lipid raftsLipid rafts are plasma membrane microdomains characterized by elevated levels of cholesterol and glycosphingolipids (1), believed to play important roles in signal transduction processes.

Biological problemMelanoma cells derived from tissues at different stages of tumor progression display different growth patterns and metastatic phenotypes. To be able to migrate and invade other tissues, signalling and adhesion molecules such as those present in rafts on the plasma membrane probably play a pivotal role.

The analytical approach : We have chosen to analyse rafts proteins by automated LC-MS/MS after proteolysis. A recent comprehensive study (2) has identified 153 integral rafts proteins as well as 91 rafts-associated proteins. Our goal was the differential analysis of raft proteins from different cell lines. Therefore a true comparative approach was necessary to go beyond a classical comparison of identified protein lists. Also, we wanted to compare simultaneously more than two samples, making an isotope labelling approach difficult.

2Introduction 1

3

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The MSIGHT (3) tool :

MSIGHT is a freeware software tool (http://www.expasy.org/MSight/) for the visualization of peptide LC-MS datasets as two-dimensional density maps. MSIGHT offers the following functionalities :

• import of data in different formats• faithful display of raw LC-MS data at variable resolution• alignment and comparisons of two or more datasets • annotation of ions analysed by CID

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Introduction 2

Cell lysis in modif MN buffer ( 25mM MES pH 6.5,150mM NaCl,1.0 mM Na2VO4, protease inhibitor cocktail (Roche), 1% Triton X-100), 20 min on ice followed by Dounce homogenisation (10 strokes).

Discontinuous Sucrose gradient : mix lysate 1:1 with sucrose 90 % and place at the bottom of tube ; 30% , 5% layers were added followed by centrifugation at 200’000 x g for 16 h at 4 C. 12 fractions collected from top to bottom, rafts-enriched fractions determined by western blot against caveolin (+ marker ) and transferrinreceptor (- marker). Fr. 2-5 were reproducibly containing rafts.

Precipitation + digestion : according to (2) ; rafts-enriched fractions were mixed with 3 vol. of MN buffer and pelleted by centrifugation (200’000 x g, 1h). Pellets were resuspended in 20 ul of 6M Urea, 2M thiourea, 10mM Hepes, pH 8.3 and subjected to ethanol/ sodium acetate precipitation. Pelleted proteins were resuspended again in a minimal volume of 6M Urea, 2M thiourea, reduced and alkylated(DTT/Iodoacetamide), diluted 4-fold with ammonium bicarbonate and digestied with trypsin at 37C for 15h. Digests were acidified and desalted on micro C18 Stage tips (Proxeon Biosystems, Denmark). Eluates were dried and resuspended in 10 ul buffer A.

LC-MS and LC-MS/MS analysis : 1ul of each sample were separated on a LC-Packings (amsterdam, Holland) Famos-Ultimate equipped with a PepMap C18 column (75um x 15 cm) run at 200 nl/min. The gradient used was : 3 min 1 6% B, 60 min 6 37% B, 3 min 37 99% B (solvent A: 0.5%; solvent B : 0.5% formic acid, 80% acetonitrile ). The LC was coupled on-line through a nanospray interface with a SCIEX QSTAR Pulsar i Qq-TOF mass spectrometer. For LC-MS/MS runs , the two most intense peaks were chosen for fragmentation and then excluded for 120 s. For differential display by MSIGHT, LC-MS runs were acquired immediately after the LC-MSMS run, with the difference that no CID was performed and every TOF MS (full scan) spectrum was accumulated for 3.0s instead than 1.0 s to increase spectral quality and decrease number of spectra to display.

4Methods : sample prep

} Rafts

Lysis,SucroseGradient

Dilute &Pellet

ReduceAlkylateTrypsinise

Analyse

5SBCl2 : from skin, RGP melanoma, non invasive, non metastatic

SK-Mel28 : from skin metastasis, VGP, invasive, metastatic

WM239 : lymph node metastasis, VGP, invasive, metastatic

Data Analysis :

MS/MS spectra

Mascot protein identification(SWISSPROT+TrEMBL)

MS spectra

Data display and alignment with MSIGHT

1) Quantify identified proteins in the three samples2) Detect differences among samples neglected in first pass analysis

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Methods : analysis

Cell lines :

6

400.0 970.0

25

80

SBCl2 ( non metastatic)

SK-Mel28 ( metastatic)

WM239 ( metastatic )

• Patterns are highly reproducible

• Intensities vary significantly (total intensity as well as relative intensities of individual proteins)

LC-MS runs as 2D maps

400.0 970.0

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m/z

min

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Score SBCL2

Score SKMel28

Score WM239A

Class (2)

ACTB_HUMAN P60709 Actin, cytoplasmic 1 (Beta-actin) 97 253 329 RA 1.0 8.6 2.5AHNK_HUMAN Q09666 Neuroblast differentiation associated protein AHNAK 167 1986 323 R 1.0 8.7 1.0CAV1_HUMAN Q03135 Caveolin-1 120 32 RA 1.0 2.0 0.3GBAK_HUMAN P08754 Guanine nucleotide-binding protein G(k), alpha subu 71 225 219 RGBB1_HUMAN P62873 Guanine nucleotide-binding protein G(I)/G(S)/G(T) b 93 138 175GBGC_HUMAN Q9UBI6 Guanine nucleotide-binding protein G(I)/G(S)/G(O) g 56 37 110 RGBI2_HUMAN P04899 Guanine nucleotide-binding protein G(i), alpha-2 280 343 379 R 1.0 3.1 1.2Q6YHK3 Q6YHK3Activated T-cell marker CD109 346 128 232 RQ6ZQN2 Q6ZQN2Hypothetical protein FLJ46846 256 1307 419 #1 1.0 13.2 1.1Q8N274 Q8N274 Hypothetical protein FLJ33834 196 835 230 #1 Q8N694 Q8N694 CD44 antigen, isoform 4 74 219 123 1.0 5.7 0.8Q8TBV2 Q8TBV2Annexin A2, isoform 2 432 385 RA 1.0 2.2 0.1Q9NYF6 Q9NYF6 Myotonic dystrophy kinase-like CDC42-binding kina 48 54 46VIME_HUMAN P08670 Vimentin 256 332 831 R 1.0 3.7 3.2

5NTD_HUMAN P21589 5'-nucleotidase precursor (EC 3.1.3.5) 572 R 1.0 0.1 0.1AMPN_HUMAN P15144 Aminopeptidase N (EC 3.4.11.2) 981 R 1.0 0.0 0.0BLK_HUMAN P51451 Tyrosine-protein kinase BLK + others 48 #2 BST2_HUMAN Q10589 Bone marrow stromal antigen 2 (BST-2) 64 RCALM_HUMAN P62158 Calmodulin (CaM) 34 GTR1_HUMAN P11166 Solute carrier family 2, facilitated glucose transporte 61 RAQ6P4R8 Q6P4R8 NFRKB protein 35Q7L0N3 Q7L0N3 NGL-1 protein 37Q9HC77 Q9HC77 Centrosomal P4.1-associated protein 40RB1A_HUMAN P62820 Ras-related protein Rab-1A (YPT1-related protein) 41 #4 1.0 2.1 0.9

#2 could be any Tyr kinase#3 Rab2, Rab3 found in (2)

R = Rafts protein ; RA = Rafts-associated (according to Foster et al (2))

GROUP 1 : Identified (MASCOT) in two out of three cell lines

GROUP 2 : Detected in the SBCl2 (non-metastatic) cell line only

MSIGHT quantif. normalized on SBCL2

#1 homolog of AHNAK

Identification vs. Quantification 1 7

Score SBCL2

Score SKMel28

Score WM239A

Class (2)

AAC1_HUMAN P12814 Alpha-actinin 1 (Alpha-actinin cytoskeletal isoform) 37ADT1_HUMAN P12235 ADP,ATP carrier protein, heart/skeletal muscle isoform T 34ECHA_HUMAN P40939 Trifunctional enzyme alpha subunit, mitochondrial 317 141 R 1.0 3.8 0.6ECHB_HUMAN P55084 Trifunctional enzyme beta subunit, mitochondrial (TP-be 103 68 R 1.0 25.2 5.4FLT1_HUMAN O75955 Flotillin-1 72 111 R 1.0 7.8 3.1FLT2_HUMAN Q14254 Flotillin-2 (Epidermal surface antigen) (ESA) 62 R 1.0 9.3 3.9GB01_HUMAN P09471 Guanine nucleotide-binding protein G(o), alpha subunit 1 136 R #1 1.0 1.7 1.4GB12_HUMAN Q03113 Guanine nucleotide-binding protein, alpha-12 subunit (G 67 R 1.0 3.7 1.0GBAS_HUMAN P63092 Guanine nucleotide-binding protein G(s), alpha subunit 86 195 R 1.0 4.5 2.5GBQ_HUMAN P50148 Guanine nucleotide-binding protein G(q), alpha subunit 37 RH2BA_HUMAN P62807 Histone H2B.a/g/h/k/l (H2B.1 A) (H2B/a) (H2B/g 38Q6IPN6 Q6IPN6 Eukaryotic translation elongation factor 1 alpha 1 45Q9Y4F1 Q9Y4F1 CDEP (FERM, RhoGEF, and pleckstrin domain protein 1, 36 R nd yes yesRALA_HUMAN P11233 Ras-related protein Ral-A 35 69 RA 1.0 5.3 3.5RAPA_HUMAN P62834 Ras-related protein Rap-1A (GTP-binding protein smg-p2 40 #2 1.0 4.6 1.6RB35_HUMAN Q15286 Ras-related protein Rab-35 (Rab-1C) (GTP-binding protei 98 R 1.0 4.0 1.6RL14_HUMAN P50914 60S ribosomal protein L14 (CAG-ISL 7) 44RL2A_HUMAN P46776 60S ribosomal protein L27a 35 33RLA2_HUMAN P05387 60S acidic ribosomal protein P2 43RRA2_HUMAN P62070 Ras-related R-Ras2 (Ras-like protein TC21) 61 R 1.0 1.8 0.8SN23_HUMAN O00161 Synaptosomal-associated protein 23 (SNAP-23) 178 R 1.0 7.3 3.3STOM_HUMAN P27105 Erythrocyte band 7 integral membrane protein (Stomatin 59 129 RA 1.0 19.6 3.0TRFM_HUMAN P08582 Melanotransferrin precursor (Melanoma-assoc. antigen p 94 1.0 0.7 4.2UBIQ_HUMAN P62988 Ubiquitin 61 1.0 6.3 2.3UBP8_HUMAN P40818 Ubiquitin carboxyl-terminal hydrolase 8 (EC 3.1.2.15) 35 1.0 6.7 2.4VA0D_HUMAN P61421 Vacuolar ATP synthase subunit d (EC 3.6.3.14) (V-ATPas 47 61VPP1_HUMAN Q93050 Vacuolar proton translocating ATPase 116 kDa subunit a 62 RAYES_HUMAN P07947 Proto-oncogene tyrosine-protein kinase YES (EC 2.7.1.1 114 R 1.0 6.0 0.8

MSIGHT quantif. normalized on SBCL2

GROUP 3 : Identified (MASCOT) only in metastatic cell lines

#2 : Rap1b found in (2)#1 : subunit 1/2 : seq. in commmon

R = Rafts protein ; RA = Rafts-associated (according to Foster et al (2))

Identification vs. Quantification 2

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539 549

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SBCl2 : non invasive SK-Mel28 : invasive WM239 : invasive

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SN23 UBIQUITIN CD109 ANX2 VIME

Extracting ion intensities

AMPN_HUMAN

0100020003000400050006000700080009000

10000

701.

9

732.

4

863

852.

1

5NTD_HUMAN

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VIME_HUMAN

0100020003000400050006000700080009000

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3

558.

33

559.

8

585.

4

662.

4

ANX2_HUMAN

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12000

14000

543.

8

556.

3

771.

99 551

615.

7

ACTB_HUMAN

02000400060008000

100001200014000160001800020000

488.

8

499.

8

566.

81 896

AHNK_HUMAN

0

10000

20000

30000

40000

50000

60000

70000

407.

23

480.

3

482.

3

504.

3

507.

8

GBI2_HUMAN

0

5000

10000

15000

20000

25000

555.

8

637.

8

460.

94 874

SBCl2 : non invasive SK-Mel28 : invasive WM239 : invasive

CD44_HUMAN

020004000

60008000

1000012000

140001600018000

481.

3

922.

5

693.

9

RB1A_HUMAN

0

500

1000

1500

2000

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3000

536.

4

658.

9

CD109

0

500

1000

1500

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2500

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3500

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451.

3

542.

3

567.

3

572.

4

871.

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CAV1_HUMAN

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550.

3

497.

3

802.

5

----G

roup

1 --

----

---G

roup

2 --

-10Quantification based on ion intensities 1

GB12_HUMAN

0100200300400500600700800900

1000

431.

2

FLT2_HUMAN

0

200

400

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1000

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600.

4

Q9Y4F1

0100200300400500600700800900

1000

690.

9

RAPA_HUMAN

0

200

400

600

800

1000

1200

1400

747.

96

RB35_HUMAN

0500

100015002000250030003500400045005000

527.

3

536.

36

STOM_HUMAN

0

1000

2000

3000

4000

5000

6000

7000

624.

36

843.

7

UBIQ_HUMAN

0

500

1000

1500

2000

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541.

3

894.

5

ECHA_HUMAN, ordered by position in protein sequence

02000400060008000

1000012000140001600018000

807.

5

544.

3

669.

8

946.

2

789.

5

602.

3

761.

4

ECHB_HUMAN

0

500

1000

1500

2000

2500

3000

3500

4000

478.

8

533.

3

618

YES_HUMAN

0100020003000400050006000700080009000

431.

8

433.

8

446.

8

512.

8

FLT1_HUMAN

0

500

1000

1500

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2500

576.

3

608.

4

SN23_HUMAN

0

500

1000

1500

2000

2500

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3500

539.

8

579.

8

GBAS_HUMAN

0

1000

2000

3000

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5000

600053

7.8

643.

9

RRA2_HUMAN

0

200

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600

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471.

3

500.

3

------

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---G

roup

3 --

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--11

SBCl2 : non invasive SK-Mel28 : invasive WM239 : invasive

Quantification based on ion intensities 2

12MSIGHT software

•LC-MS runs can be faithfully represented as 2D maps ; images can be aligned and compared

•Extracted peak intensities provide good semi-quantitative measures of protein amounts

•Limitations : manual peak extraction and quantification slow, inaccurate process

Quantification Results

• highly reproducible patterns matching and alignment is possible

• Sampling by MS/MS is incomplete in such a complex mixture

• Proteins identified only in one sample can be quantified in all samples by pattern matching and extracting ion intensities

• ID scores are not an accurate measures of protein presence/ absence nor quantity, at least when analyses are not redundant and sampling is incomplete.

• Problem : relative amounts of Rafts proteins in different samples vary significantly difficult to find a “most common ratio” for normalisation.

Conclusions 1

References1) Zajchowski LD, et al, Eur J Biochem. 2002 Feb;269(3):737-522) Foster, LJ et al. PNAS May 13, 2003 , vol. 100 no. 10 , 5813–58183) P. M. Palagi, et al, MSight: a 2-D LC-MS image analysis software, Proteomics, in press

13Rafts results

•Most proteins, even those that were identified only in one sample, appear to be present in rafts from all three cell lines

•Two proteins (ECHA, ECHB) were found to be upregulated in metastatic cells. However so far we failed to find metastatic – specific proteins. Three proteins ( AMPN, 5NTD, ANX2) were found to be downregulated in metastatic cells compared with non-metastatic ones.

Perspectives

• This is a preliminary study with small amounts of proteins ; scale-up is in progress !

• MSIGHT quantification will allow the simultaneous comparison of more than 3 cell lines ; automatic peak detection and quantification functionalities will drastically accelerate the analysis process

• Peptides differentially expressed but not analysed by CID can be located and listed for a subsequent targeted analysis

Conclusions & Perspectives