Letter pubs.acs.org/ac
High-Sensitivity N‑Glycoproteomic Analysis of Mouse Brain Tissue by Protein Extraction with a Mild Detergent of N‑Dodecyl β‑D-Maltoside Jing Liu,†,‡ Fangjun Wang,*,† Jiawei Mao,†,‡ Zhang Zhang,†,‡ Zheyi Liu,†,‡ Guang Huang,†,‡ Kai Cheng,†,‡ and Hanfa Zou*,† †
Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences (CAS), Dalian 116023, China ‡ University of Chinese Academy of Sciences, Beijing 100049, China S Supporting Information *
ABSTRACT: N-dodecyl β-D-maltoside (DDM), a mild detergent with the ability to maintain the enzyme activity and solubilize hydrophobic proteins without changing their structures, was applied for N-glycoproteomic analysis of minute protein sample from mouse brain tissue. After combining with the capillary-based glycoproteomic reactor, 281 N-glycosylation sites were successfully characterized from 50 μg of mouse brain tissue, which was 110% higher at least than those obtained by conventional strategies.
P
buffers with urea and conventional detergents (SDS, Triton X100, and RapiGest SF) for minute protein sample extraction. Furthermore, as DDM is compatible with trypsin digestion, the tedious detergent removal steps were avoided and the protein sample can be online purified by a reversed phase trap column before liquid chromatography coupled with tandem mass spectrometry (LC−MS/MS) analysis. Combining this new protein sample extraction strategy with our previous reported capillary-based N-glycoproteomic reactor,18 we have characterized 281 N-glycosylation sites corresponding to 170 Nglycoproteins from only 50 μg of mouse brain tissue, which is about 2 times the numbers identified by using conventional sample preparation strategies. We first optimized the concentration of DDM within the lysis buffer. Mouse brain tissues (about 5−10 mg for each) were lysed using the lysis buffers with 0, 0.1%, 0.25%, 0.5%, 1%, and 2% DDM at the ratio of 10 μL buffer/1 mg tissue, respectively. The protein concentration of each lysate was 5.0 ± 0.2, 6.5 ± 1.0, 7.2 ± 1.0, 7.5 ± 1.1, 9.0 ± 0.5, and 8.8 ± 0.6 mg/ mL, respectively (Supporting Information, Figure S1 and Table S1). The protein concentration first increased along with the increase of DDM concentration and reached to a maximum after the DDM concentration was ≥1%. Therefore, lysis buffer with 1% DDM was utilized in our following experiments. Then, we investigated the performance of protein extraction by using
rotein extraction, including the lysis of cells or tissues, release of proteins from different cell compartments, and solubilization of proteins into extraction buffer, is the first step of protein sample preparation.1,2 Usually, the transmembrane proteins are too hydrophobic to extract into the aqueous solution, and strong detergents and chaotropes are necessary to disrupt the membranes and increase the protein extraction efficiency.3−5 Strong detergents, such as sodium dodecyl sulfate (SDS),6 are most widely utilized in the extraction of proteins with high hydrophobicity, such as the glycosylated transmembrane proteins. However, the strong detergents would significantly decrease the efficiency of enzyme digestion,7,8 chromatography separation, and mass spectrometry detection. Thus, removing the detergent by protein precipitation with organic solvents, dialysis, gel filtration, or the recently developed filter-aided sample preparation (FASP)6 is necessary before protein digestion and MS detection. Unfortunately, the process of detergent removal usually induces significant protein sample loss due to the buffer exchange and redissolving steps, which compromises the sensitivity, reliability, and accuracy of proteomic analysis especially for the post translational modifications (PTMs) analysis of a minute amount of protein samples.3,9 N-Dodecyl β-D-maltoside (DDM) is a nonionic detergent composed of a lauryl hydrophobic chain and a maltose hydrophilic part. It is a mild detergent with the ability to maintain the enzyme activity,10 purify membrane proteins,11,12 and solubilize hydrophobic proteins without changing their structures.13−17 Here, we demonstrated the lysis buffer with DDM and urea exhibited comparable performance to the © 2015 American Chemical Society
Received: December 18, 2014 Accepted: February 3, 2015 Published: February 3, 2015 2054
DOI: 10.1021/ac504700t Anal. Chem. 2015, 87, 2054−2057
Letter
Analytical Chemistry seven different lysis buffers. Briefly, TEAB buffer, TEAB buffer containing 8 M urea, TEAB buffer containing 8 M urea and 1% detergent (Triton X-100, RapiGest SF or SDS), TEAB buffer containing 4% SDS,1,6 and TEAB buffer containing 8 M urea and 1% DDM were utilized. Finally, the protein concentration of each lysate was 2.6 ± 0.3 (TEAB buffer), 4.9 ± 0.2 (8 M urea buffer), 8.6 ± 1.0 (8 M urea buffer with 1% Triton X-100), 9.0 ± 1.2 (8 M urea buffer with 1% RapiGest SF), 9.1 ± 0.7 (8 M urea buffer with 1% SDS), 8.8 ± 0.3 (4% SDS buffer), and 8.9 ± 0.4 (8 M urea buffer with 1% DDM) mg/mL, respectively (Figure 1 and Supporting Information, Table S2). Obviously,
Table 1. Sequence Coverages and Numbers of Unique Peptides Identified from 1 μg of BSA Digests with Different Concentration of DDM DDM concn (mg/mL)
sequence coverage (%)
unique peptides
0 0.1 0.2 0.5 1 2 5 10
77.3 78.3 77.3 78.7 76.4 78.4 74.0 74.6
55 52 51 52 53 51 50 52
the relative standard deviation (RSD) for peptides retention time were all
