Native protein mapping and visualization of protein interactions in human plasma high-density lipoprotein by 2-DE and LC-MS/MS
A human plasma sample was subjected to non-denaturing micro 2-DE and a gel area (5 mm X 18 mm) which includes high-density lipoprotein (HDL) was cut into 1 mm X 1 mm squares, then the proteins in the 90 gel pieces were analyzed by in-gel trypsin digestion and nano-LC-MS/MS. Grid-cutting of the gel was employed to; 1) ensure the total analysis of the proteins in the area, 2) standardize the conditions of analysis by LC-MS/MS, 3) reconstruct the protein distribution patterns from the label-free quantification data. Totally 154 proteins (excluding keratins) were assigned in the 90 gel pieces and the quantity distribution of each was reconstructed as a color density pattern (a native protein map). The map of apolipoprotein (Apo) A-I showed a wide apparent mass distribution characteristic to HDL and was compared with the maps of the other 153 proteins. Eleven proteins showed maps of wide distribution that overlapped with the map of Apo A-I, and all have been reported to be the components of HDL. Further, seven minor proteins associated with HDL were detected at the gel positions of high Apo A-I quantity. These results for the first time visualized the localization of HDL apolipoproteins on a non-denaturing 2-DE gel and strongly suggested their interactions.
Sample Processing Protocol
Non-denaturing micro 2-DE: An aliquot of 2-uL of a human plasma sample from an apparently healthy individual (37 y, female) was subjected to IEF in the absence of denaturants employing agarose column gel (1.4 mm id × 35 mm) which contained 5% v/v Pharmalyte pH 3-10 and 1% w/v agarose. IEF was run at 0.12 mA/gel CC until a voltage of 300 V was reached (15 min) and continued at 300 V CV for 35 min. The IEF apparatus was chilled in an ice-water bath during the focusing. The focused gel was transferred onto the top of a polyacrylamide micro slab gel (4.2-17.85% T linear gradient, 5% C，38 X 38 X 1 mm3), on which a 100-µL aliquot of a 0.01 M Tris-0.076 M glycine buffer (pH 8.3) was added beforehand. The second-dimensional electrophoresis was run using a 0.05 M Tris-0.38 M glycine buffer (pH 8.3) at 10 mA/gel constant current, and continued until the spots of albumin, visualized by the binding of bromophenol blue added in the electrophoresis buffer, moved to about 15 mm from the gel bottom (~50 min). The micro slab gel was stained in 0.1% w/v CBB in 50% methanol/7% acetic acid v/v for 15 min, destained in 20% methanol/7% acetic acid for 120 min (one change), rehydrated in 7% acetic acid and stored in 7% acetic acid at 4 ºC until use. Gel grid cutting: A gel area which has been assigned to contain HDL by immunochemical staining was subjected to grid cutting, as shown in the gel pattern. The 5 mm X 18 mm grid area was cut into 1 mm X 1 mm squares providing ninety 1-mm3 gel pieces. Destaining, reduction/alkylation and in-gel digestion: Each gel piece was put in a 0.5-mL tube and equilibrated with a solution of 100 mM NH4HCO3 (50 uL) for 10 min and a 50-uL aliquot of acetonitrile was further added in the tube. The tube was left for 10 min to remove CBB and the gel piece was washed with water (50 uL) for 10 min and dehydrated with acetonitrile (20 uL) for 10 min. The solution was discarded and the gel piece was dried in a vacuum evaporator-centrifuge for 10 min. The gel was rehydrated with a solution of 10 mM DTT in 100 mM NH4HCO3 (20 uL) and kept at 56 C with mild shaking for 1 h. The tube was cooled down to room temperature and the solution was discarded, a 20-uL aliquot of 50 mM iodoacetamide in 100 mM NH4HCO3 was added, and the alkylation of cysteine residues was achieved by keeping the tube in darkness for 45 min with mild shaking (room temp.). The gel piece was washed in 50 uL 100 mM NH4HCO3 for 10 min, dehydrated with acetonitrile for 10 min, dried in vacuum, rehydrated in a 10-uL aliquot of trypsin solution (10 ng/uL in 50 mM NH4HCO3), and trypsin digestion was done at 37 C overnight (~13 h). The solution was taken out to a new tube, the gel was further subjected to peptide extraction with a 10-uL aliquot of 0.1 % TFA/50% acetonitrile (v/v) for 20 min with sonication, and the solution was also transferred to the tube. The pooled solution was dried by vacuum centrifugation and then reconstituted with a 10-uL aliquot of 1% v/v formic acid-2% v/v acetonitrile. Nano-UPLC: Prior to nano-UPLC−MSE analysis, each tryptic peptide sample (10 µL) was added a 2 µL-aliquot of the Enolase Digest Standard (240 fmol/µL) prepared in 1% v/v formic acid-2% v/v acetonitrile. Nano-LC separation of the peptides was performed with a nanoACQUITY system (Waters) equipped with a Symmetry 5 µm C18, 180 µm X 20 mm trap column and a UPLC 1.7 µm BEH130 C18, 75 µm X 100 mm analytical reverse phase column (both Waters). Mobile phase A was 0.1% v/v formic acid in water whilst mobile phase B was 0.1% formic acid in acetonitrile. Each sample, 5 µL full loop injection, was initially transferred with 0.5% mobile phase B (99.5% mobile phase A) to the trap column at a flow rate of 5 µL/min for 3 min. The peptides were then eluted from the trap column to the analytical column and separated at a flow rate of 300 nL/min with a gradient as follows: 2% B for 2 min, ramp to 50% B over 18 min, ramp to 85% B over 1 min, hold at 85% B for 5 min, then ramp to 2% B over 1 min. The column temperature was maintained at 35 ºC. The lock mass compound, leucine enkephalin (m/z 556.2771) or [Glu1]-Fibrinopeptide B (m/z 785.8427), was delivered at 0.05 µL/min and at a concentration of 1 ng/µL to the reference sprayer of the NanoLockSpray source of the mass spectrometer via the sample pump of the MS apparatus. Mass spectrometry: On-line mass spectrometric measurement of the nano-UPLC-separated tryptic peptides was performed using a Q-TOF mass spectrometer (Synapt G2-S HDMS, Waters) equipped with a nano-ESI source. The mass spectrometer was operated in positive and resolution mode for all the measurement. LC-MS/MS data were collected with the instrument operating in MSE mode (data-independent mode). Data were acquired collecting spectra every 0.6 sec with alternating low (6 eV, 0.3 sec) and elevated (ramp from 20 to 40 eV, 0.3 sec) energy over a m/z 350-2000 range. The lock mass channel was sampled every 60 sec.
Data Processing Protocol
The nano-LC-MSE data were processed with ProteinLynx Global SERVER (PLGS) ver. 2.5.2 (Waters), using the following parameters: database, UniProtKB homo sapiens complete proteome dataset (canonical sequences only, 20251 entries, 2013-05-29); peptide and fragment tolerance, both automatic (typically <10 ppm and <20 ppm, respectively); maximum of missed trypsin cleavage, 1; maximum protein mass, 600 kDa; fixed modification, carbamidomethylation at Cys; variable modifications, oxidation at Met; false positive rate, 4%. The criteria of protein identification during PLGS processing were set as; at least one peptide matches per protein, at least three fragment ion matches per peptide and at least seven fragment ion matches per protein. Protein quantities were calculated by PLGS referring to the quantity of the internal standard (tryptic peptides of ENO1_YEAST). For each of the 90 gel piece, there are an “S(square no.).raw.zip” file (the raw LC-MSE data) and an “S(square no.). Ident result.zip” file (the search results output by PLGS, including three .CSV files corresponding to protein, peptide and fragment-level identification results, respectively.) Also, an Excel file combining the protein-level identification results of all the spots is provided, named as "ya.jin HDL-Grid S1-90 summary.xlxs". Please note, all the identification results are original and have not been depleted of TRYP_PIG, ENO1_YEAST (standard), protein entries with scores lower than 100 and/or with only one peptide identified and/or without quantification data.
Jin Y, Bu S, Zhang J, Yuan Q, Manabe T, Tan W. Native protein mapping and visualization of protein interactions in the area of human plasma high-density lipoprotein by combining non-denaturing micro 2-DE and quantitative LC-MS/MS. Electrophoresis. 2014 Mar 26 PubMed: 24668886