Supporting Information

Carbon Nanotubes (2,5-dihydroxybenzoylhydrazine) Derivative as pH adjustable enriching reagent and Matrix for MALDI Analysis of Trace Peptides

Shi-fang Ren, Yin-long Guo*

(Shanghai Mass Spectrometry Center,Shanghai Institute of Organic Chemistry, ChineseAcademy of Sciences, Shanghai 200032, PRC)

Supporting Information

S1. Synthesis details

S2. TGA curves of the samples

S3. MALDI-TOF mass spectra of the peptide mixtures with or without the functionalized CNT-mediate enrichment

S1. Details of Synthesis Procedure for functionalized CNTs

Typical procedure (see the scheme below) of synthesizing functionalized CNTs is described as follows, including oxidation of the CNT, activation of the carboxyl moieties with thionyl chloride and subsequent reaction with hydrazine.

1) Synthesis of acid-functionalized CNTs

A raw sample of nanotubes (600mg) was first refluxed in 40ml of 2.6 M nitric acid for 20 hours. The resultant suspension was then diluted with 200ml water, and larger CNTs were collected on a 100-nm pore filter membrane (type VCTP; Millipore) and washed with water. Dry at 100 ºC.

2) Synthesis of 2,5-dihydroxybenzoyl hydrazine

2,5-dihydroxybenzoate was firstly prepared from 10mmol of 2,5-dihydroxybenzoyl acid which was refluxed in methanol for 3 hours catalyzed by concentrated sulfuric acid. After removal of the solvent under reduced pressure, the product was washed with water and dried over sodium sulfate.

Then to a stirred solution of 2,5-dihydroxybenzoate (10 mmol) in methanol (10 mL) was added hydrazine hydrate (85%, 10 mmol)). The mixture was stirred at room temperature for 4 h and then concentrated in vacuo. The crude product was recrystallized from methanol and dichloromethane to give 2,5-dihydroxybenzoyl hydrazine with 95% yield.

3) Synthesis of CNT 2,5-dihydroxybenzoyl hydrazine derivative

The CNT hydrazine derivative was prepared from 20mg of acid-functionalized CNT placed in a dry 100-mL flask to which 20mL of thionyl chloride was added, and the mixture was stirred for 12 h. The reaction mixture was filtered through a 100-nm pore filter membrane, and then obtained precipitate was flushed with a large amount of dry acetone. The solid precipitate was dried in air and then placed in a 100-mL flask containing 5g 2,5-dihydroxybenzoyl hydrazine in 40mL dry THF. The mixture was refluxed for 12 h and then dried in vacuo. The product was poured into a large amount of ethanol and sonicated for another 10 min followed by filtration and dry.

S2. TGA curves of the samples

Figure S2TGA thermograms of (A) pristine CNT, (B) CNT 2,5-dihydroxybenzoyl hydrazine derivative derivative.

S3. MALDI-TOF mass spectra of the peptide mixtures with or without the functionalized CNT-mediate enrichment

Figure S3 MALDI-TOF mass spectra of the peptide mixtures: a) 1 pg/μL prepared without enrichment, and the peptides mixture after enrichment by functionalized CNTs in peptide mixture solution of b) 0.1 pg/μL, c) 0.01 pg/μL. CHCA was used as matrix.

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