Detection of matrix metalloproteinase active forms in complex proteomes: evaluation of affinity versus photoaffinity capture.
Various attempts to detect matrix metalloproteinase (MMP) active forms from complex proteomes, based on the use of specific photoactivatable affinity probes, have up to now failed. To overcome this failure, an affinity approach has been evaluated as an alternative to the photoaffinity one. For this purpose, two probes were synthesized to interact specifically with the active site of MMPs and allow isolation of MMP/probe complexes on magnetic beads through a biotin linker. Using phosphinic peptide chemistry, we prepared an affinity probe displaying picomolar potency toward several MMPs, and a related photoaffinity probe incorporating a photoactivatable azido group exhibiting subnanomolar affinity toward these targets. By a combination of silver-staining detection and MALDI peptide mass fingerprints, a systematic comparison was made of both strategies in terms of hMMP-12 and hMMP-8 recovery and identification when present in mixtures of different complexity. The results obtained show that the affinity protocol is superior to the photoaffinity strategy in terms of quantity of captured MMPs and number of MMP tryptic fragments detected in MALDI-MS. The specificity and efficiency of the affinity capture protocol developed in this study allowed easy, fast, and unambiguous detection by MALDI-MS of three hMMPs (2, 8, and 12), from a single affinity capture experiment, when added (10-36 ng of MMPs) to a tumor extract (10 microg). Thus, the tools and approaches reported should enable us to progress in the detection of endogenous active forms of MMPs in complex proteomes, an important objective with many diagnostic applications.
Bregant
Sarah
S
CEA, iBiTecS, Service d'Ingénierie Moléculaire des Protéines (SIMOPRO), Bat 152, CE-Saclay Gif/Yvette, F-91191, France.
Huillet
Céline
C
Devel
Laurent
L
Dabert-Gay
Anne-Sophie
AS
Beau
Fabrice
F
Thai
Robert
R
Czarny
Bertrand
B
Yiotakis
Athanasios
A
Dive
Vincent
V
D000345:Affinity Labels / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D045744:Cell Line, Tumor; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D053508:Matrix Metalloproteinase 12 / Q000737:chemistry / Q000378:metabolism; D020778:Matrix Metalloproteinase 2 / Q000737:chemistry / Q000378:metabolism; D020784:Matrix Metalloproteinase 8 / Q000737:chemistry / Q000378:metabolism; D020782:Matrix Metalloproteinases / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D015394:Molecular Structure; D019920:Photoaffinity Labels / Q000737:chemistry; D020543:Proteome / Q000737:chemistry / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000345:Affinity Labels; D019920:Photoaffinity Labels; D020543:Proteome; D020782:Matrix Metalloproteinases; D020778:Matrix Metalloproteinase 2; D020784:Matrix Metalloproteinase 8; D053508:Matrix Metalloproteinase 12