Difference in Mass Analysis Using Labeled Lysines (DIMAL-K): A New, Efficient Proteomic Quantification Method Applied to the Analysis of Astrocytic Secretomes

Joël Poncet,Joël Bockaert,Nathalie Galéotti,Eric Mauger,Patrick Jouin,Emmanuelle Demey,Nicolas Delcourt,Philippe Marin

doi:10.1074/mcp.m500040-mcp200

Joël Poncet, Joël Bockaert + Show 6 more

Open Access

https://doi.org/10.1074/mcp.m500040-mcp200

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Abstract

Here we describe an original strategy for unbiased quantification of protein expression called difference in mass analysis using labeled lysine (K) (DIMAL-K). DIMAL-K is based on the differential predigestion labeling of lysine residues in complex protein mixtures. The method is relevant for proteomic analysis by two-dimensional electrophoresis and MALDI-TOF mass spectrometry. Protein labeling on lysine residues uses two closely related chemical reagents, S-methyl thioacetimidate and S-methyl thiopropionimidate. Using protein standards, we demonstrated that 1) the chemical labeling was quantitative, specific, and rapid; 2) the differentially labeled proteins co-migrated on two-dimensional gels; and 3) the identification by mass fingerprinting and the relative quantification of the proteins were possible from a single MALDI-TOF mass spectrum. The power of the method was tested by comparing and quantifying the secretion of proteins in normal and proinflammatory astrocytic secretomes (20 microg). We showed that DIMAL-K was more sensitive and accurate than densitometric image analysis and allowed the detection and quantification of novel proteins.

Highlights

We describe an original strategy for unbiased quantification of protein expression called difference in mass analysis using labeled lysine (K) (DIMAL-K)
Quantification of Variations in Astrocytic Protein Secretion Induced by a Proinflammatory Treatment by Using the DIMAL-K Strategy—Previously we have identified the major proteins secreted by astrocytes, the major cell population in the mammalian central nervous system, using a proteomic approach combining the separation of proteins recovered from astrocyte culture-conditioned medium by 2-D electrophoresis and their identification by MALDI-TOF MS [24]
This procedure, which we call DIMAL-K, is based on the differential labeling of lysine residues on entire proteins and is relevant for proteomic approaches based on separation of proteins by 2-D electrophoresis and their identification by MALDI-TOF mass spectrometry

Summary

EXPERIMENTAL PROCEDURES

Materials—S-Methyl thioacetimidate and S-methyl thiopropionimidate were synthesized according to Beardsley and Reilly [22]. Preparation of Astrocyte-conditioned Media—Cells were washed six times with the serum-free culture medium. Q-TOF Mass Spectrometry Analysis—10 ␮g of standard protein labeled with either S-methyl thioacetimidate or S-methyl thiopropionimidate were solubilized in 20 ␮l of 1% formic acid, and 2 ␮l were transferred in a tip emitter (Protana, Odense, Denmark). MALDI-TOF Mass Spectrometry and Protein Identification—Proteins of interest were excised and digested in-gel using trypsin (sequencing grade, Promega, Charbonnieres, France) as described previously [27]. Peptides were selected in the mass range of 900 – 4,000 Da. Peptide mass fingerprint identification of labeled proteins was performed by searching against the Swiss-Prot Databases using an in-house Mascot software package (Matrix Science) with Arg-C enzyme specificity and no trypsin missed cleavages. These scripts were written to run on various platforms (Win, MacOSX, Linux, and UNIX) and can be obtained freely on request (eric.mauger@ ccipe.cnrs.fr)

RESULTS

Sequence coverage of labeled protein

DISCUSSION

Tryptic peptides