The presence of highly abundant proteins can interfere with the resolution and sensitivity of the proteomic techniques used to analyse the serum profiles. For this reason, serum samples were depleted by immunoaffinity chromatography using a Multiple Affinity Removal System (MARS) column (4.6 mm ID × 100 mm, Agilent Technologies Inc., CA, USA) containing antibodies against the six most abundant serum proteins: albumin, IgG, IgA, transferrin, haptoglobin and alpha-1-antitrypsin. The depletion is expected to remove about 88-92% of the total protein content. Removal was performed according to the recommendations of the manufacturer. Briefly, 200 μL of diluted sample were injected in a Beckman System Gold HPLC (Beckman Coulter, Fullerton, CA, USA). The low-abundance protein fractions eluted were aliquoted and stored at −20°C until analysis. The QC was also depleted following the same procedure.
Mars column
Manufactured by Agilent Technologies
Sourced in United States
The MARS) column is a chromatography column designed for the separation and purification of small molecules. It utilizes a reversed-phase packing material to facilitate the separation process. The core function of the MARS) column is to provide efficient and reliable separation of chemical compounds in a variety of analytical and preparative applications.
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Lab products found in correlation
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Beckman system gold hplc, by Beckman Coulter (1 mentions)
Sequencing grade modified trypsin, by Promega (1 mentions)
Sep pak c18 column, by Waters Corporation (1 mentions)
Ltq orbitrap velos, by Thermo Fisher Scientific (1 mentions)
1200 series hplc, by Agilent Technologies (1 mentions)
4 protocols using mars column
1
Serum Protein Depletion for Proteomic Analysis
2
Urine Protein Depletion Protocol
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Equal amount of proteins from the concentrated urine samples from healthy individuals and patients from each group was pooled and processed for depletion as per manufacturer's instructions. Urine samples were pooled to avoid the heterogeneity of samples between the patients and ignore the individual differences. We pooled the samples to differentiating each group of patients rather than individuals. Briefly, human six immunoaffinity multiple affinity removal system (MARS column, 4.6 mm × 100 mm, Agilent Technologies, Santa Clara, CA, USA) was connected to a high-performance liquid chromatography system and equilibrated with Agilent buffer-A. We loaded 4 mg of urinary protein from each group onto the column to deplete six abundant proteins (albumin, IgG, IgA, antitrypsin, transferrin, and haptoglobin). Flow-through fractions were collected, concentrated and desalted using 5 kDa molecular weight cut-off spin column (Agilent, Palo Alto, CA). We determined the protein concentration using Bradford assay.
3
Quantitative Proteomics Using SID-MRM-MS
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Multiple affinity removal system (MARS) column and a 24-well setup and a 24 cm, pH 3–10 IPG strip were purchased from Agilent Technologies (Santa Clara, CA, United States). Sequencing grade modified trypsin was from Promega (Madison, WI, United States).
Nine isotope-labeled peptides were synthesized as internal standards (ISs) for SID-MRM-MS (AnyGen Co., Gwangju, Korea): NLSEAQL*R, TEDTIFLR, YGAATF*TR, ISASAEEL*R, GWVTDGFSSL*K, LIQGAP*TIR, AEDAPGVPL*R, VSVTL*SGK and GFQALGDAADI*R for Calmodulin-like protein 5 (CALML5), Alpha-1-acid glycoprotein 2 (ORM2), Pregnancy zone protein (PZP), Apolipoprotein A-IV (APOA4), Apolipoprotein C-III (APOC3), Insulin-like growth factor-binding protein 2 (IGFBP2), Mucin-5AC (MUC5AC), Pancreatic triacylglycerol lipase (PNLIP) and Metalloproteinase inhibitor 1 (TIMP1), respectively. * represents the amino acid labeled with the 13C15N heavy isotope.
Nine isotope-labeled peptides were synthesized as internal standards (ISs) for SID-MRM-MS (AnyGen Co., Gwangju, Korea): NLSEAQL*R, TEDTIFLR, YGAATF*TR, ISASAEEL*R, GWVTDGFSSL*K, LIQGAP*TIR, AEDAPGVPL*R, VSVTL*SGK and GFQALGDAADI*R for Calmodulin-like protein 5 (CALML5), Alpha-1-acid glycoprotein 2 (ORM2), Pregnancy zone protein (PZP), Apolipoprotein A-IV (APOA4), Apolipoprotein C-III (APOC3), Insulin-like growth factor-binding protein 2 (IGFBP2), Mucin-5AC (MUC5AC), Pancreatic triacylglycerol lipase (PNLIP) and Metalloproteinase inhibitor 1 (TIMP1), respectively. * represents the amino acid labeled with the 13C15N heavy isotope.
4
Gambian Children Plasma Proteomics
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Plasma samples from 140 Gambian children aged 2 to 59 months were used for proteomic studies. Samples were obtained from patients included in clusters 124, 125, 126 and 132. Individual samples (5 µl of plasma) were pooled into 3 different groups (~55 µl of plasma per batch) in each cluster category: 35 individual samples were randomly divided into three groups of 11, 12 and 12 samples (see Fig. 2c ). Pooled plasma samples were depleted of the top 14 highly-abundant plasma proteins with a multiple affinity removal (MARS) column (Agilent, UK) using high-performance liquid-chromatography (HPLC) 1200 series (Agilent, UK). Proteins from depleted plasma were precipitated with trichloroacetic acid and quantified using a colorimetric assay (BCA Protein assay, Thermo Scientific, US) and further separated by size using SDS-PAGE and bands were cut and digested with trypsin. Peptide digests were purified using Sep-Pak C18 columns (Waters, Milford, MA). The nano-LC system (final rate 0.3 μl/min) was coupled to LTQ-Orbitrap Velos (Thermo) and searched against the human proteome with a false-discovery rate of 1% calculated from target-decoy hits and relative (label-free) quantification was based on normalized spectral index quantitation (SINQ)35 (link).
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