Bruker Daltonics

Abstract

Isotope-Coded Protein Label (ICPL [1,2]) is known as an accurate protein labeling strategy for quantitative proteomics which enables protein pre-fractionation and therefore provides reduction of complexity, detection of protein isoforms, posttranslational modifications, and investigations of enzymatic reactions. Such an investigation is demonstrated in a workflow using SDS-PAGE for protein pre-fractionation, a Bruker Ion Trap mass spectrometer, and ProteinScapeTM for quantitative analysis.

Introduction

Proteolytic modification of components of the extracellular milieu by metalloproteinases plays important roles in the regulation of multiple cellular and physiological processes and pathological conditions. Exploring the substrate repertoire of metalloprotenaises is required to unveil the mechanisms involved in their biological function. ADAMTS1 (A Disintegrin and Metalloprotease domain with Thrombospondin motifs 1), is a secreted enzyme of the ADAMTS family of proteases, which is related to angiogenesis, inflammation, and cancer [3]. We describe a proteomic screening for ADAMTS1 substrates by analyzing the protein profiles obtained from cultures of transfected cells overexpressing the

Application Note # LCMS-56

ICPL Labeling in Functional Proteomics Experiments: Substrate Identification of the Extracellular Protease ADAMTS1 using SDS-PAGE LC-MS/MS

protease as compared to parental cells. The secreted proteins from the two cell lines were modified with two different isotopomers of the non isobaric ICPL duplex label, mixed and quantitatively compared in a SDS-PAGE LC-MS/MS workflow. Seven proteins were identified as putative substrates of ADAMTS1.

Experimental

Conditioned media from 293T cells (human embryonic kidney), parental and overexpressing the secreted protease ADAMTS1, were collected. Proteins were concentrated by UF, then subjected to TCA-acetone precipitation, resuspended in guanidinium chloride buffer and quantified. 100 micrograms of each sample were then labeled with ICPL. The ICPL reagent is shown in Figure 1. Parental cell proteins were labeled with the heavy isotopomer of ICPL doublex, and ADAMTS1 overexpressing cells with the light isotopomer of ICPL. The mixture of the two labeled samples was separated by SDS-PAGE. The gel lane was cut into twenty fractions and subjected to in-gel trypsin digestion. Each digest was analyzed by nano RPLC-MS/MS on a Bruker Ion Trap system. Quantitation of relative protein abundances was performed based on the chromatographic signal intensities of labeled peptide pairs, using ProteinScape as integrated software platform for quantitative LC-MS/MS workflows. The applied workflow is visualised in Figure 2.

Results

The ICPL based 1D-SDS LC-MS workflow allowed an efficient quantitative comparison of the analyzed proteomes. A total of 827 proteins were identified on the basis of 2001 peptide IDs. Quantitative data were obtained for 511 proteins (61%), based on 1254 labeled peptide IDs. Labeling was highly efficient, as almost no peptides with non-derivatized lysine residues were observed (>99%).In agreement with previous experiments using DIGE [2], basal lamina Nidogen 1 and 2, were identified as putative substrates of the ADAMTS1 protease. With the ICPL strategy provided here, the ID of new putative substrates was achieved. These results are summarized in Table 1.Using a quantitative top-down analysis strategy (SDS-PAGE) proteolytic degradation can be detected: A substrate protein occurs in several bands and shows in the intact protein band a different regulation than in the bands of the proteolytic fragments. Figure 3 shows Nidogen-1 as example, which was identified in several fractions: In fraction 2 we observe the intact protein, in fraction 3 the high mass proteolytic fragment PF1 and in fraction 10 the low mass proteolytic fragment PF 2. The abundance of the intact Nidogen-1 is about 2 times lower in the ADAMTS1 overexpressing cells, while in the fractions where its proteolytic fragments are

Figure 1: ICPL Duplex labeling chemistry for quantitative proteomics

Figure 2: Experimental Workflow. ICPL permits protein pre-fractionation and quantification via MS spectra. Regulated peptides are preferred precursors in AutoMS(n) measurements, which lead to protein identification with Mascot.

ICPL – Isotope Coded Protein Label, Duplex

ICPL workflow

XX

NXX

X XO

NO

O

O

12C/13C-Nicotinoyloxy-succinimide

Light : X = 12C

Heavy: X = 13C

m = 6 units

ICPL – Isotope Coded Protein Label, Duplex

-NH2 labeling: Lysine , N-terminal

1D SDS-PAGE ICPL JC 12_1-B,1_01_1086.d: TIC +All

0

2

4

6

9x10Intens.

20 40 60 80 100 120 140 160 Time [min]

nanoRP-LCESI-IT

- 20 slices-Trypsin digestion

582.3

673.8

749.6

873.4

928.6986.0

1051.8

1124.3

1320.8

749.6

673.8

+MS, 87.2min #1983

0.0

0.5

1.0

1.5

2.0

2.57x10

Intens.

400 600 800 1000 1200 1400 1600 m/z

MS

450.1478.1

553.2

596.7653.2

743.2 800.8

882.6

975.8

1004.3

1047.31105.4

1192.31304.2

+MS2(749.5), 87.3min #1984

0

1

2

3

46x10

Intens.

400 600 800 1000 1200 1400 m/z

375.2

448.1

503.2

539.1 610.2

655.7

689.2

790.3

841.3

885.3

950.3

1041.3 1136.4

+MS2(674.1), 87.3min #1985

0.0

0.5

1.0

1.5

6x10Intens.

200 400 600 800 1000 1200 m/z

MS2 MS2

ICPLQuantitation

Protein identification

(Mascot)

ProteinScape

RP

ICPL labeling

observed, their abundance is increased 2 times compared to the control cells. The analysis of the band containing the Nidogen-1 proteolytic fragment PF1 is presented in Figure 4. By using ProteinScape for data processing, the complete information of the proteomic experiment, e.g. meta information of the experiment, the gel, the MS and MS/MS data as well as the derived identification and quantitation is stored and linked at a central place and can be easily retrieved with the aid of different views and queries.

Table 1: Putative substrates of ADAMTS1 and IDed proteolytic fragments. Fragments exhibit opposite regulation as parent proteins. The results are in agreement with previous experiments using DIGE [3].

Quantitation of Nidogen-1

ProteinFL: full length

PF: proteo-lytic fragment

Mapped region (total aminoacids)

Obs. Mw

(kDa)

Total Peptides

Labeled Peptides

ADAMTS1/ Control ratio

(L/H ICPL) (StdDev)

ADAMTS1/Control

ratio (DIGE)

ADAMTS1 FL 105 78 72 >20 10

Nidogen 1 FL 124-1247 (1247) 180 9 2 -1.85 (0.04) -1.61

Nidogen 1 PF1 424-1247 (1247) 116 21 6 2.46 (0.74) 3.25

Nidogen 1 PF2 124-176 (1247) 40 18 7 1.88 (0.40)

Nidogen 2 PF1 309-968 (1375) 180 10 2 2.19 (0.40)

Nidogen 2 PF2 91-171 (1375) 38 4 1 2.05 1.86

Fibulin-1 PF1 396-576 (703) 38 10 6 3.24 (0.37)

Fibulin-1 PF2 182-619 (703) 55 8 2 3.32 (1.03)

Beta-amyloid protein precursor (APP)

PF1 103-516 (770) 55 5 3 1.33 (0.42)

Beta-amyloid protein precursor (APP)

PF2 117-535 (770) 80 9 3 1.24 (0.16)

Nucleobindin-1 FL 54-461 (461) 55-60 20 10 -1.44 (0.10)

Nucleobindin-1 PF1 97-310 (461) 40 19 10 1.71 (0.40)

Nucleobindin-1 PF2 179-377 (461) 25 10 5 3.14 (0.78)

Insulin-like growth factor binding protein 2

FL 45-320 (328) 36 12 5 -3.56 (0.10)

Insulin-like growth factor binding protein 2

PF1 45-240 (328) 15 7 2 5.42 (0.73)

Insulin-like growth factor binding protein 2

PF2 284-304 (328) 9 3 2 1.67 (0.02)

Legumain FL 45-403 (433) 55 7 4-2.88 (0.08)

Legumain PF1 318-433 (433) 20 8 5 4.69 (0.95)

6

20

37

55

100

29

120201

1D SDS-PAGE

FL 180 kDaL/H = -1.85

PF1 120 kDaL/H = 2.46

PF2 35 kDaL/H = 1.88

1 MLASSSRIRA AWTRALLLPL LLAGPVGCLS RQELFPFGPG QGDLELEDGD 51 DFVSPALELS GALRFYDRSD IDAVYVTTNG IIATSEPPAK ESHPGLFPPT 101 FGAVAPFLAD LDTTDGLGKV YYREDLSPSI TQRAAECVHR GFPEISFQPS 151 SAVVVTWESV APYQGPSRDP DQKGKRNTFQ AVLASSDSSS YAIFLYPEDG 201 LQFHTTFSKK ENNQVPAVVA FSQGSVGFLW KSNGAYNIFA NDRESIENLA 251 KSSNSGQQGV WVFEIGSPAT TNGVVPADVI LGTEDGAEYD DEDEDYDLAT 301 TRLGLEDVGT TPFSYKALRR GGADTYSVPS VLSPRRAATE RPLGPPTERT 351 RSFQLAVETF HQQHPQVIDV DEVEETGVVF SYNTDSRQTC ANNRHQCSVH 401 AECRDYATGF CCSCVAGYTG NGRQCVAEGS PQRVNGKVKG IFVGSSQVPR 451 IVFENTDLHS YVVMNHGRSY TAISTIPETV GYSLLPLAPV GGIIGWMFAV 501 EQDGFKNGFS ITGGEFTRQA EVTFVGHPGN LVIKQRFSGI DEHGHLTIDT 551 ELEGRVPQIP FGSSVHIEPY TELYHYSTSV ITSSSTREYT VTEPERDGAS

601 PSRIYTYQWR QTITFQECVH DDSRPALPST QQLSVDSVFV LYNQEEKILR 651 YALSNSIGPV REGSPDALQN PCYIGTHGCD TNAACRPGPR TQFTCECSIG 701 FRGDGRTCYD IDECSEQPSV CGSHTICNNH PGTFRCECVE GYQFSDEGTC 751 VAVVDQRPIN YCETGLHNCD IPQRAQCIYT GGSSYTCSCL PGFSGDGQAC 8 01 QDVDECQPSR CHPDAFCYNT PGSFTCQCKP GYQGDGFRCV PGEVEKTRCQ 851 HEREHILGAA GATDPQRPIP PGLFVPECDA HGHYAPTQCH GSTGYCWCVD 901 RDGREVEGTR TRPGMTPPCL STVAPPIHQG PAVPTAVIPL PPGTHLLFAQ 951 TGKIERLPLE GNTMRKTEAK AFLHVPAKVI IGLAFDCVDK MVYWTDITEP 1001 SIGRASLHGG EPTTIIRQDL GSPEGIAVDH LGRNIFWTDS NLDRIEVAKL 1051 DGTQRRVLFE TDLVNPRGIV TDSVRGNLYW TDWNRDNPKI ETSYMDGTNR 1101 RILVQDDLGL PNGLTFDAFS SQLCWVDAGT NRAECLNPSQ PSRRKALEGL 1151 QYPFAVTSYG KNLYFTDWKM NSVVALDLAI SKETDAFQPH KQTRLYGITT 1201 ALSQCPQGHN YCSVNNGGCT HLCLATPGSR TCRCPDNTLG VDCIERK

PF2

PF1

Figure 3: Identification and quantitation of Nidogen-1 in different bands of the 1D gel. Compared to the control, Nidogen-1 is down regulated in the fraction containing the intact protein (FL), while it is up regulated in the fractions containing the proteolytic fragments (PF1 and PF2).

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References

[1] A. Schmidt, J. Kellermann, F. Lottspeich,

Proteomics 5 (2005), 4-15.

[2] Bruker Daltonics Application Note #MT-78, ICPL: A new

quantitative mass spectrometric method for proteomics.

[3] F. Canals, N. Colome, C. Ferrer, MdelC. Plaza-Calonge,

J.C. Rodriguez-Manzaneque, Proteomics 6 Suppl 1 (2006), 28-35.

Authors

Francesc Canals1, Nuria Colomé1, Joan J. Bech-Serra1, Juan Carlos Rodríguez-Manzaneque2

1 Vall d’Hebron Institute of Oncology (VHIO), Vall d’Hebron University Hospital, Barcelona2 Pfizer-University of Granada-Andalusian Government Center for Genomics and Oncological Research (GENYO), Granada

Wolfgang Jabs, Markus Lubeck, Carsten Baessmann, Detlev SuckauBruker Daltonik GmbH, Bremen

Keywords

Cancer

ICPL labeling

Metalloproteinase

Quantitative Analysis

Functional Proteomics

Instrumentation & Software

Bruker Ion Trap mass spectrometer

ProteinScape

For research use only. Not for use in diagnostic procedures.

ProteinScape stores the complete information of the experiment

Figure 4: Analysis of the experiment using ProteinScape. The screenshot shows the Mascot Search result for the fraction containing the Nidogen-1 proteolytic fragment PF1. The software allows storage and linkage of the complete information gathered during a proteomic experiment at a central place. The gel separation, identification and quantitation of proteins and peptides of the selected band are displayed.

Conclusion

ICPL labeling chemistry in combination with SDS-PAGE LC-MS/MS allowed the identification of new putative substrates of metalloproteinase ADAMTS1.The proteolytic fragments were assigned based on their MW in SDS-PAGE, their significant up regulation in comparison to the down regulation of the full length sequences, and their reduced coverage of the parent protein sequence.