Secondary-Ion Mass Spectrometry of Genetically Encoded Targets

Secondary ion mass spectrometry (SIMS) is generally used in imaging the isotopic composition of various materials. It is becoming increasingly popular in biology, especially for investigations of cellular metabolism. However, individual proteins are difficult to identify in SIMS, which limits the ability of this technology to study individual compartments or protein complexes. We present a method for specific protein isotopic and fluorescence labeling (SPILL), based on a novel click reaction with isotopic probes. Using this method, we added 19F-enriched labels to different proteins, and visualized them by NanoSIMS and fluorescence microscopy. The 19F signal allowed the precise visualization of the protein of interest, with minimal background, and enabled correlative studies of protein distribution and cellular metabolism or composition. SPILL can be applied to biological systems suitable for click chemistry, which include most cell-culture systems, as well as small model organisms.

Constructs. The vector pCMV t-RNA-PylRS WT was a gift from Dr. Edward Lemke (EMBL, Heidelberg) and was used as previously described. [5] For the fluorescent protein constructs from Figure S1, plasmids containing GFP (SNAP-25, syntaxin 13) and YFP (syntaxin 1) at the C-terminus were used. In the rest of this work, pN1 vectors that contain an Ochre stop codon (TAA) immediately after the Amber codon, and no FP (fluorescent protein) tag, were employed.
The plasmids encoding for SNAP-25B (pEYFP-C1) and syntaxin 1A (pEYFP-N1) were a gift from Professor Thorsten Lang (LIMES Institute, University of Bonn, Germany). SNAP-25 was subcloned into a pEGFP-N1 (Clontech) vector. Both SNAP-25 (in pEGFP-N1) and syntaxin 1 (in pEYFP-N1) constructs were subjected to site-directed mutagenesis. Their respective variants lacking the fluorescent protein moieties (pN1) were obtained by enzymatic restriction with appropriate enzymes, followed by the introduction of Ochre stop codons after the coding sequence.
The plasmid for syntaxin 13 (pET28a) was a gift from Professor Reinhard Jahn (Max Planck Institute for Biophysical Chemistry, Göttingen, Germany). The syntaxin 13 gene was directly subcloned into pEGFP-N1 and pN1 vectors containing Amber stop codons in the linker region in front of the GFP sequence (pEGFP-N1) or in the residual linker region after the enzymatic excision of GFP (pN1).

Solid Phase Peptide Synthesis
Manual SPPS of peptide 3 was performed under microwave irradiation starting with a 50 µmol scale on a Fmoc-Gly-preloaded Sieber amide resin (0.76 mmol/g). The resin was swollen in a BD syringe (Becton Dickinson, Fraga, Spain) for 2 h in DMF, and by washing with DMF (3×), DCM (3×) and NMP (3×), before starting the coupling cycle by microwave PRK incorporation was carried out by co-transfecting cells with two vectors: pCMV tRNA-Pyl RS WT (encoding for the bioorthogonal aminoacyl-tRNA-synthetase/tRNA pair) [5] and the vectors encoding for the proteins of interest (SNAP-25, syntaxin 1, and syntaxin 13).
Transfection using Lipofectamine 2000 Reagent (Life Technologies) was performed by first equilibrating for 5 min plasmids and Lipofectamine with Opti-MEM (Gibco) in separate Eppendorf tubes, and subsequently mixing the two and incubating them for 20 min. The mixture was applied on the cells, which were incubated at 37 °C for 18 h. Refer to Figure S5 for an overview of the experimental timeline. The medium was exchanged to normal DMEM 2 h prior to fixation.
The samples were embedded in LR White resin (London Resin Company Ltd, Berkshire, England) as previously described [6] . The LR White-embedded samples where cut using an EM UC6 ultramicrotome (Leica Microsystems) into 200 nm samples that were then mounted on silicone wafers.

Confocal microscopy.
Multichannel confocal images were taken with a Leica TCS SP5 microscope (Leica Microsystems) using a 100× 1.4 N.A. HCX PL APO CS oil objective (Leica). To visualize the fluorophores, the following excitation lines were employed: an argon laser at a wavelength of 488 nm for Cy2, and a helium-neon laser at 633 nm for Star635. An AOTF filter (Leica) was used to select appropriate emission intervals. Signal detection was performed using photomultiplier tubes. Images were acquired at 7.5× zoom by scanning sequentially line-by-line at 1,000 Hz the two channels. Final images have a 1024×1024 pixels format with pixel size of 20.21×20.21 nm (for a 20.68×20.68 µm scanned area).

Secondary Ion Mass Spectrometry (SIMS). NanoSIMS measurements were performed using a NanoSIMS 50L instrument (Cameca, Gennevilliers, France), installed at TUM
Freising. Samples were sputtered using a fine focused Cs + primary ion beam at 16 keV energy. The resulting secondary ions were separated by mass and detected by electron multipliers. Measurements were performed with a primary current of ~1-2 pA, at an estimated lateral resolution of ~100 nm. The mass resolving power was set to reach a reliable separation of the potential isobars. Before the measurement, a pre-sputtering with a high primary current was performed for the implantation of Cs + ions promoting the ionization efficiency. Sample areas of 20×20 µm were scanned with 512×512 pixel and a 10 ms dwell-time per pixel.
Unless otherwise stated, figures represent single plane measurements. When multiple planes were measured, image accumulation was done using a Matlab routine. To image the distribution of relevant species, the following ions were detected: 19 F -, 12 C 14 N -, 12 C 15 N -, referred through the manuscript as 19 F, 14 N and 15 N, respectively. For presentation purposes, the images were binned using a 2x2 binning procedure. Fluorescence in the DAPI channel was detected using a 377/50 excitation filter, 409 long pass beam splitter and 447/60 emission filter. For the green channel (GFP or YFP/FITC), the following filters were used: 480/40 HQ excitation filter, 505 LP Q beam splitter, and 527/30 HQ emission filter. In the red channel, Abberior Star635P fluorescence was detected using the 620/60 HQ excitation filter, the 660 LP Q beam splitter, and the 700/75 HQ emission filter.
All filters were produced by AHF (Tübingen, Germany). Data analysis. All analyses were performed using self-written routines in Matlab (the Mathworks Inc, Natick, MA). The fluorescence and SIMS images were overlaid and corrected for drifts and for rotational misalignments. Circular regions of 9 pixels in diameter were then collected manually in the different images. The average fluorescence intensity, or the average isotopic counts, were determined, and plotted in Figures 3 and 4. In Figure 3B the ratio between 19 F and 14 N was used, rather than the raw levels of 19 F, since it is less subjected to experimental variations in NanoSIMS imaging. We would also like to point out that the analysis of ratios, rather than absolute levels, is a routine procedure in SIMS.
Statistics. The student's t-test was used to assess the difference between the 19 F/ 14 N ratios in transfected versus non-transfected cells ( Figure 3B).    [2,3] (2) was synthesized by a copper(II) catalayzed diazo transfer reaction with previously prepared 1H-imidazole-1-sulfonyl azide hydrochloride [1,2] (1) as an azide transfer reagent. (B) Fmoc-Lys(N 3 )-OH (2) was incorporated by manual solid phase peptide synthesis (SPPS) under microwave irradiation, following the Fmoc-protocol on Sieber amide resin. 2,3,4,5,6-Pentafluorobenzoic acid was introduced following standard coupling conditions. [7] Cleavage from the resin and simultaneous removal of protecting groups yielded crude peptide 3, which was purified by HPLC and characterized by high-resolution mass spectrometry. (C) Labeling of peptide 3 with Star635 NHS ester was accomplished under light exclusion in solution to yield SK155. This compound was purified by HPLC and characterized by high-resolution mass spectrometry.