Improving Peptides de novo Sequencing for MALDI-MSI by in situ N-terminal Derivatization Julien Franck1; Mohamed El-Ayed1; Jonathan Stauber1; Maxence Wisztorski1; Pierre Olivier Schmit2; Rabah Boukheroub3; Isabelle Fournier1; Michel Salzet1 1MALDI
Imaging Team, FRE CNRS 2933, USTL, Villeneuve d'Ascq, France; 2Bruker Daltonique, Wissembourg, France 3Institut de Recherche Interdisciplinaire (IRI), 59655 Villeneuve d’Ascq, France
Overview
Results
Purpose:
10 years old FFE tissue
Improve identification of peptides resulting from in situ enzymatic digestion of tissue section
m/z 945.6 m/z 1068.2
Methods: Trypsic in situ digestion and matrix deposition by robot (ChIP-1000, Shimadzu Co., Kyoto, Japan) Direct on-tissue derivatization
m/z 945.6
Results: N-terminal derivatization on both frozen and FFPE tissue section Sulfonated peptides gives a better higher MASCOT score than their homologues without derivatization
ChIP-1000, Shimadzu
Ultraflex II, Bruker
m/z 772.2 + m/z 1340.6
Introduction •MALDI direct analysis and imaging of tissue sections show continuously increased performances in term of different class of detected molecules, images spatial resolution, number of detected compounds, sensitivity and applications. Besides obtaining compounds biodistribution possible identification of compounds is of prime importance. However, in situ characterization of peptides/proteins is still a critical step for MALDI-MSI. In this context, on tissue N-terminal derivatizations represent a good alternative for improving in situ de novo sequencing. •Structural information on proteins is a crucial point for identification of proteins mapped in MALDI-MSI and thus comprehension of biological processes. However, retrieving such information’s directly from the tissue without using classical extraction step would increase MALDI-MSI potential. Protein identification can be difficult especially if studied organism genome data are not available. De novo sequencing is then required but can reveal difficulties depending on the studied peptide. Moreover, previous studies reveal that direct MS/MS of peptides from tissues is not successful. Ions seem to be much more stables when directly desorbed from tissue. However, using a strategy where MS/MS is performed after enzymatic digestion already improved identification. Even better identification can be obtained especially for de novo sequencing by using N-terminal derivatizations. •In these studies, different N-terminal derivatizations were tested directly on tissues. Best results were obtained with sulfonation reagents like SPITC1. Studies reveal that N-terminal derivatization is very difficult such as but works very well after a first step of enzymatic digestion.
Fresh Frozen tissue
Figure 1: Principle of trypsin and matrix deposition with piezoelectric-bases chemical inkjet printer on (a) FFPE tissue, (b) Frozen tissue (b)
(a)
cncn
(c)
Figure 2: Direct analysis of tissue section (a) after enzymatic digestion with trypsin, (b) after derivatization with a sulfonic reagent, (c) after derivatization with 4-SPITC (a)
(b)
Methods Tissue preparation : Frozen tissue section (10µm) were obtained with cryostat and washed in cloroform2 10 years old FFPE tissue section (10 µm) were obtained with cryotome. MALDI imaging Enzymatic digestions were performed directly on tissue sections using trypsin (0,050 µg/µL) in water. A piezoelectric-based chemical ink-jet printer (ChIP-1000, Shimadzu Co., Kyoto, Japan) was used for deposition of both the trypsin and DHB (10mgmL) in MeOH/TFA 0,1% as matrix2. MALDI images and MS/MS were performed on a MALDI-TOF/TOF Ultraflex II (Bruker Daltonics, Bremmen). Images were reconstructed using MALDI fleximaging Tool (Bruker Daltonics) MALDI direct analysis and N-terminal derivatization N-terminal derivatizations were performed with various reagents like 4-SPITC. Derivatization reagents (10 mg/mL) deposition was first performed using a micropipette in Tris-HCl buffer (pH = 8,5) for optimization before tests with the chemical ink-jet printer. Tissue section are then incubate for 1 hour at 55°c.
Figure 3: (a) MS/MS spectra of m/z 1139.699, (b) MS/MS spectra of m/z 1139.69 after sulfonation with sulfonic reagent (m/z 1523.67)
Conclusions Enzymatic digestion followed by N-terminal derivatization can be performed for both frozen or formalin fixed and paraffin embedded conserved tissue. Much better fragmentation yields of fragmentation were obtained after derivatization as also orientated fragmentations as expected allowing for easy interpretation of MS/MS data and peptide sequence determination. Prior to sulfonation, the ion m/z 1139,699 could be identified for HRDTGILDSIGR as a tryptic digest fragment of Myelin. A MASCOT-ion score of 61 was achieved. After sulfonation, a nearly complete y-type ion series were identified (y1-10) resulted in a much higher score of 80 Compatibility of derivatization with FFPE conserved tissues open a the door of a great potential for identifying biomarkers directly form tissue conserved in hospital tissue banks. Supported by grants from Centre National de la Recherche Scientifique (CNRS), Ministère de la Recherche et des Technologies (MRT, ACI jeunes Chercheurs to I. Fournier), Institut National du Cancer (INCA, to I. Fournier), collaboration agreement between Bruker Daltonics and Laboratoire de Neuroimmunologie des Annelides, collaboration with Shimadzu for the ChIP1000
More informations about MALDI Imaging team of Annelid’s Neuroimmunology Laboratory: http://www.maldi-imaging.com
Corresponding author:
[email protected] 