LC-MS/MS analysis of apical and basolateral plasma membranes of rat renal collecting duct cells

Abstract

We used biotinylation and streptavidin affinity chromatography to label and enrich proteins from apical and basolateral membranes of rat kidney inner medullary collecting ducts (IMCDs) prior to LC-MS/MS protein identification. To enrich apical membrane proteins and bound peripheral membrane proteins, IMCDs were perfusion-labeled with primary amine-reactive biotinylation reagents at 2 degrees C using a double barreled pipette. The perfusion-biotinylated proteins and proteins bound to them were isolated with CaptAvidin-agarose beads, separated with SDS-PAGE, and sliced into continuous gel pieces for LC-MS/MS protein identification (LTQ, Thermo Electron Corp.). 17 integral and glycosylphosphatidylinositol (GPI)-linked membrane proteins and 44 non-integral membrane proteins were identified. Immunofluorescence confocal microscopy confirmed ACVRL1, H(+)/K(+)-ATPase alpha1, NHE2, and TauT expression in the IMCDs. Basement membrane and basolateral membrane proteins were biotinylated via incubation of IMCD suspensions with biotinylation reagents on ice. 23 integral and GPI-linked membrane proteins and 134 non-integral membrane proteins were identified. Analyses of non-integral membrane proteins preferentially identified in the perfusion-biotinylated and not in the incubation-biotinylated IMCDs revealed protein kinases, scaffold proteins, SNARE proteins, motor proteins, small GTP-binding proteins, and related proteins that may be involved in vasopressin-stimulated AQP2, UT-A1, and ENaC regulation. A World Wide Web-accessible database was constructed of 222 membrane proteins (integral and GPI-linked) from this study and prior studies.

Fig. 1. Apical surface biotinylation via retrograde perfusion of IMCDs

A, a rat kidney medulla was excised and placed on a porous support that allows fluid drainage and in between filter papers that moisturize the tissue. Apical membrane proteins were labeled with biotin via perfusing the IMCD lumens using a custom-made double barreled pipette in a cold room (2 °C) to inhibit endocytosis. One barrel delivered paraformal-dehyde to fix the membrane lipids before the other barrel delivered sulfo-NHS-LC-biotin or sulfo-NHS-SS-biotin to label the apical membrane proteins. The fixation of lipids prior to biotinylation of proteins was necessary to ensure apical protein biotinylation. Blue and red food dyes were used in the perfusates to visualize fluid change in the IMCDs. A single barreled pipette at the top of the perfusion pipette supplied Tris buffer to moisturize the tissue and to quench the biotinylation reagent if backflow occurred. B, a picture shows the perfusion setup in the cold room. C, a fluorescence micrograph shows biotinylation occurring throughout the perfused IMCD cells without prior fixation revealed by streptavidin-FITC that stains biotin in green. The IMCD marker AQP2 staining and the DAPI nuclear staining are shown in red and blue, respectively. D, a fluorescence micrograph shows restricted apical membrane biotinylation in the perfused IMCDs with prior fixation.

Fig. 2

A, a flow chart summarizing the isolation, preparation, and identification of proteins in the fixed perfusion-biotinylated IMCDs. B, a silver-stained image showing the isolation processes of biotinylated proteins and their associated proteins in the fixed perfusion-biotinylated IMCDs. The total membrane fraction (lane T) was prepared and bound to the CaptAvidin-agarose beads. After removal of the unbound (U) and the non-specifically bound proteins in three washing buffers (W_1–3), the biotinylated proteins and their associated proteins were eluted (E) from the CaptAvidin-agarose beads. C, a silver-stained image showing how the biotinylated proteins and their associated proteins were separated with SDS-PAGE and sliced into 16 pieces (numbers on the left) to reduce sample complexity prior to preparation for LC-MS/MS protein identification. Lane M, molecular mass markers.

Fig. 3. Confocal immunofluorescence microscopy confirming the expression of proteins identified from IMCDs

Activin A receptor type II-like I, potassium-transporting ATPase α1, sodium/hydrogen exchanger 2, and sodium- and chloride-dependent taurine transporter in the IMCDs are visualized with secondary antibodies conjugated to FITC or Alexa488 (green). These proteins co-localize with IMCD marker protein aquaporin 2 (red, Alexa568), indicating their expression in the IMCDs. The nuclei are stained with DAPI (blue).

Fig. 4. RT-PCR confirmation of mRNA expression of one-peptide identifications in the IMCDs

PCR with prior RT reaction (lane +) using AQP2, an IMCD marker protein, specific primers served as a positive control. PCR without reverse transcription reaction (lane −) served as the negative controls. Parentheses indicate sample sources: FPB, fixed perfusion-biotinylated IMCDs; FIB, fixed incubation-biotinylated IMCDs; NPB, non-fixed perfusion-biotinylated IMCDs; NIB, non-fixed incubation-biotinylated IMCDs. ACE, angiotensin-converting enzyme; APC, adenomatous polyposis coli protein; CA4, carbonic anhydrase 4; CD40L, CD40 ligand; CLIC4, chloride intracellular channel protein 4; ERC2, ERC protein 2; FZD1, frizzled 1 precursor; GPR64, G-protein coupled receptor 64 precursor; GRIN2D, glutamate (N-methyl-D-aspartate) receptor subunit ε 4 precursor; HCN1, potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 1; Kv4.3, potassium voltage-gated channel subfamily D member 3; LPL, lipoprotein lipase; LRP4, low density lipoprotein receptor-related protein 4; MAPK12, mitogen-activated protein kinase 12; MCT2, monocarboxylate transporter 2; PalmT, palmitoyl-transferase ZDHHC7; PIK3-C2γ, phosphatidylinositol-4-phosphate 3-kinase C2 domain-containing γ polypeptide; PIK3-Cβ, phospha-tidylinositol-4,5-bisphosphate 3-kinase catalytic subunit β isoform; PKA-βC, cAMP-dependent protein kinase, β-catalytic subunit; PODXL, podocalyxin; ROMK, ATP-sensitive inward rectifier potassium channel 1; S1P-R, sphingosine-1-phosphate receptor Edg-8; SCN5A, sodium channel protein type 5 α subunit; SNAP29, synap-tosomal-associated protein 29; THIK-1, potassium channel subfamily K member 13; TYRO3, tyrosine-protein kinase receptor TYRO3; VAPA, vesicle-associated membrane protein-associated protein A; VKγC, vitamin K-dependent gamma-carboxylase; WASPIP, Wiskott-Aldrich syndrome protein interacting protein.

Fig. 5. Basolateral surface biotinylation of isolated IMCD suspensions

A, an immunoblot shows that the IMCD suspensions (I) are enriched in the IMCD marker protein AQP2 and depleted of the non-IMCD marker protein AQP1 compared with the whole inner medulla homogenate (W) and the non-IMCD suspensions (N). B and C, fluorescence micrographs show that biotinylation occurs at the basement membrane and the basolateral membrane of the isolated IMCD segments in suspensions without (B) or with fixation (C) as revealed by streptavidin-FITC that stains the sites of biotinylation in green. AQP2 antibody identifies IMCD segments (red, Alexa568) and DAPI stains nuclei (blue).

Fig. 6. A Venn diagram summarizing the non-integral membrane proteins (non-MPs) identified in the fixed perfusion-biotinylated (FPB) IMCD and those identified in the fixed incubation-biotinylated (FIB) IMCD suspension

Numbers indicate protein identifications. Percentages indicate non-overlapping identifications in a particular sample preparation.

Fig. 7. Examples of integral and GPI-linked membrane proteins identified in the fixed (top) and the non-fixed (bottom) perfusion-biotinylated IMCDs

Type I and II membrane proteins have one single transmembrane span with N′ or C′ terminus facing the extracellular space, respectively. Type III membrane proteins contain multiple membrane-spanning topology. Some membrane proteins are anchored to the membrane via a GPI anchor. LPL, lipoprotein lipase; LRP4, low density lipoprotein receptor-related protein 4; ANPEP, aminopeptidase N.