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Microfluidic ion stripper for removal of trifluoroacetic acid from mobile phases used in HILIC-MS of intact proteins

ABSTRACT: Trifluoroacetic acid (TFA) is commonly used as mobile phase additive to improve retention and peak shape characteristics in hydrophilic interaction liquid chromatography (HILIC) of intact proteins. However, when using electrospray ionization-mass spectrometry (ESI-MS) detection, TFA causes ionization suppression and adduct formation leading to reduced analyte sensitivity. To overcome the latter issues, in this work we describe a membrane-based microfluidic chip with with multiple parallel channels (Microfluidic Esi Mobile Phase Ion Stripper, uEMPIS) for the selective post-column removal of TFA anions from HILIC eluents up to flow rates of 0.2 mL/min. An anion-exchange membrane was used to physically separate the column effluent from a stripper flow solution (running countercurrent) comprising acetonitrile, formic acid, and propionic acid. The exchange of ions via diffusion based on concentration gradient allowed the ion-exchange removal of TFA ions used during HILIC analysis of model proteins using mobile phases containing 0.1% TFA (13 mM). Separation selectivity and efficiency were maintained (with minor band broadening effects) while increasing the signal intensity and areas by improving ionization and reducing TFA adduct formation.

INSTRUMENT(S): maXis

ORGANISM(S): Reference Proteins

SUBMITTER: Gargano Andrea

PROVIDER: MSV000086798 | MassIVE | Wed Feb 03 00:33:00 GMT 2021

REPOSITORIES: MassIVE

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Project description:Bacteria was grown at 30C in 3 different conditions, i.e. SYN (syngas and minimal medium- ATCC no 1789), AC (0.3% acetate in minimal medium- ATCC no 1789) and TSB (tryptic soy broth). After harvesting by centrifugation, O. carboxidovorans pellets (1g) were lysed in 100 mM Tris-Cl pH 8.0, 2% Triton X-100, 2.6 mg/ml sodium azide, 8 mM PMSF by sonication on ice (4 pulses of 15 s duration each). For each condition of growth 4 samples (1g pellets) were separately treated (i.e. lysed and processed further). The supernatants were treated with 50% cold TCA, and the precipitated protein washed with acetone. The pellets were resuspended in solubilization buffer (7M urea, 20 mM tris-Cl, pH 8.0, 5 mM EDTA, 5 mM MgCl2, 4% CHAPS), and protein concentration was determined using the Plus One 2-D Quant Kit (Amersham) following the manufacturers instructions. Protein samples from each treatment were stored at -80 C. One hundred micrograms of each protein sample was resuspended in 0.1 M ammonium bicarbonate, 5% HPLC grade ACN, reduced in 5 mM DTT (65 C, 5 min), alkylated in 10 mM iodoacetamide (30 C, 30 min), and then trypsin digested until there was no visible pellet (1:50 w/w 37 C, 16 h). Peptides were desalted using a peptide microtrap (Michrom BioResources, Auburn, CA) and eluted using a 0.1% TFA, 95% ACN solution. Desalted peptides were dried in a vacuum centrifuge and resuspended in 20 ?l of 0.1% formic acid. Peptides were separated by strong cation exchange (SCX) liquid chromatography (LC) followed by reverse phase (RP) LC coupled directly in line with electrospray ionization (ESI) tandem mass spectrometry (MS/MS). 2DLC ESI MS/MS was done exactly as described (1). All searches were done using TurboSEQUEST (Bioworks Browser 3.2; Thermo Electron). Mass spectra and tandem mass spectra were searched against all annotated proteins from the strain OM5 including all the annotated plasmid-encoded proteins. Cysteine carbamidomethylation and methionine oxidation (single and double) were included in the search strategy. We used the reverse database functionality in Bioworks 3.2 and searched MS2 data against a reversed OM5 database using identical search criteria.

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Microfluidic ion stripper for removal of trifluoroacetic acid from mobile phases used in HILIC-MS of intact proteins

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