Generation of the Mouse SuperMix Column
Mouse plasma (CD-1 Mouse Plasma w/ EDTA-K3, male, 55.8 mg/mL) was obtained from Charles River Laboratories International, Inc. (Wilmington, MA). A custom SuperMix column was generated by GenWay Biotech (San Diego, CA) (now commercially available from Sigma-Aldrich, St. Louis, MO). A mouse plasma sample was initially depleted of its 7 most abundant proteins (serum albumin, IgG, fibrinogen, α1-antitrypsin, transferrin, haptoglobin and IgM) by using a Seppro™
mouse IgY7 LC10 column (Genway Biotech). Approximately 100 mg of the IgY7-depleted plasma were provided to GenWay Biotech as an antigen mixture for producing the custom mouse SuperMix column. The generation of the SuperMix LC5 column (12.7 × 39.5 mm) was similar to that previously described for producing the human SuperMix column 18
Tandem Separations of Mouse Plasma by IgY7 and SuperMix Columns
The HPLC setup18
used for plasma depletion was modified to allow the two separation steps to be applied in-series on-line to make the overall experiments automatic and less labor-intensive. Briefly, IgY7 LC10 and SuperMix LC5 columns were connected in tandem on an Agilent 1100 series HPLC system to allow sample flow through both columns following injection. A six-port valve (Agilent, Santa Clara, CA) was introduced between the two columns (). Positions 3 and 5 were plugged to prohibit back-flush towards the waste or columns during valve switching. And a “T” connector was introduced to allow the liquid stream from IgY7 or IgY7-Supermix columns to be directed towards the UV detector and subsequently towards the fraction collector. This configuration allowed collection of the flow-through and bound portions in different fractions.
Figure 1 The tandem IgY7-SuperMix separation strategy. (A) IgY7 and SuperMix columns are connected to ports 1 and 2 of a six-port valve. Port 1 is connected to port 2 when mouse plasma sample is injected into the columns. The flow-through is collected as SuperMix (more ...)
For each injection, 80 μL of mouse plasma was diluted 12-fold in dilution buffer (10 mM Tris-HCl, 150 mM NaCl, pH 7.4), filtered through 0.45 μm cellulose acetate centrifuge tube filters (Sigma-Aldrich), and injected into the tandem IgY7 and SuperMix separation system to capture the high and moderate abundance proteins, respectively. The flow-through was collected as SuperMix flow-through. Elution buffer (100 mM glycine, pH 2.5) was used to collect the IgY7 bound fraction (valve connected to collecting plate, ) and SuperMix bound fraction (valve connected to Supermix column, ). The separation scheme consisted of sample loading and washing with dilution buffer (0.5 mL/min, 45 min), IgY7 eluting (2 mL/min, 15 min), SuperMix eluting (1 mL/min, 20 min), neutralization (100 mM Tris-HCl, pH 8.0) (1 ml/min, 20 min), and re-equilibration with dilution buffer (1 mL/min, 20 min), with a total cycle time of 120 min. The separation parameters for loading, washing, and elution were chosen based on manufacturer’s instructions on IgY-LC10 and LC5 columns. To collect the IgY7 flow-through portion, the valve between the two columns was switched from SuperMix column to collecting plate () when plasma sample was injected, so that the sample will be collected without passing through the SuperMix column. All collected fractions were concentrated in Amicon Ultra-15 concentrators (3-kDa nominal molecular mass limit; Millipore, Billerica, MA) followed by buffer exchange to 50 mM NH4HCO3, pH 8.0. Protein concentration was determined using a BCA protein assay from Pierce (Rockford, IL).
To demonstrate the ability for measuring differential protein abundances, the following six non-mouse standard proteins were spiked into three 80 μL mouse plasma samples at 0.8, 4, and 20 μg/ml, respectively: 1) Bovine Carbonic Anhydrase 2 (BCA), 2) Bovine Beta-Lactoglobulin (BBL), 3) E. coli Beta-Galactosidase (EBG), 4) Bovine Alpha-Lactalbumin (BAL), 5) Equine Skeletal Muscle Myoglobin (ESMM), and 6) Chicken Ovalbumin (CO). The tandem separation was performed as described above. The SuperMix flow-through and bound fractions were collected separately for further analysis.
Protein samples were denatured using 50% 2,2,2-Trifluoroethanol (TFE) in 50 mM NH4HCO3 buffer (pH 8.0) at 60 °C for 2 h and reduced by 2 mM DTT at 37 °C for 1 h. The resulting protein mixture was diluted 6-fold with 50 mM NH4HCO3, and digested by sequencing grade modified porcine trypsin (Promega, Madison, WI) for 3 h at 37 °C in a trypsin:protein ratio of 1:50 (w/w). The final peptide concentration of the supernatant was determined using BCA protein assay. All samples were stored at −80 °C until further analysis.
Strong Cation Exchange (SCX) Fractionation
SCX fractionation of digested peptides was performed using an Agilent 1100 series HPLC system at a flow rate of 200 μL/min. 150 μg tryptic peptides from either the IgY7 flow-through or SuperMix flow-through fractions were resuspended in buffer A (25% acetonitrile, 10 mM ammonium formate, pH 3.0) and loaded onto a 2.1 × 200 mm (5 μm, 300 Å) Polysulfoethyl A LC column (PolyLC, Columbia, MD) preceded by a 2.1 × 10 mm guard column. The fractionation scheme consisted of 10 min at 100% buffer A, a 40 min linear gradient from 0 to 50% buffer B (25% acetonitrile, 500 mM ammonium formate, pH 6.8), a 10 min linear gradient from 50 to 100% buffer B, and 20 min at 100% buffer B. 25 fractions were collected for each sample via an automated fraction collector based on the UV trace. Each fraction was lyophilized prior to MS analysis.
Reverse-phase Capillary LC-MS/MS Analysis
Peptides were analyzed using a custom-built automated four-column high pressure capillary LC system coupled on-line to either an LTQ or LTQ-Orbitrap mass spectrometer (Thermo Scientific, San Jose, CA) via a nanoelectrospray ionization interface manufactured in-house. The reverse-phase capillary column was prepared by slurry-packing 3-μm Jupiter C18 bonded particles (Phenomenex, Torrence, CA) into a 65-cm-long, 75-μm-inner diameter fused silica capillary (Polymicro Technologies, Phoenix, AZ). After loading 1 μg of peptides onto the column, the mobile phase was held at 100% A (0.1% formic acid) for 20 min, followed by a linear gradient from 0 to 70% buffer B (0.1% formic acid in 90% acetonitrile) over 85 min with a flow rate ~500 nL/min. Each full MS scan (m/z 400–2000) was followed by collision-induced MS/MS scans (normalized collision energy setting of 35%) for the 10 most abundant ions. The dynamic exclusion duration was set to 1 min, the heated capillary was maintained at 200 °C, and the ESI voltage was held at 2.2 kV.
LC-MS/MS raw data were converted into .dta files using Extract_MSn (version 3.0) in Bioworks Cluster 3.2 (Thermo Scientific), and the SEQUEST algorithm (version 27, revision 12) was used to independently search all the MS/MS spectra against the mouse International Protein Index (IPI) database that had 51,489 total protein entries (version 3.35, released October 24, 2007). The false discovery rate (FDR) was estimated based on decoy-database searching methodology 19, 20
. Search parameters and filtering criteria were applied to limit the FDR at the unique peptide level to <5%, as described previously18
. Identified proteins were grouped to a non-redundant protein list using ProteinProphet™
software, after which one protein IPI number was randomly selected to represent each corresponding protein group that consisted of a number of database entries. Only those proteins or protein groups with two or more unique peptide identifications were considered as confident identifications.