In situ molecular imaging of proteins in tissues using mass spectrometry.

Knowledge of the location and abundance of proteins in different cellular regions of tissue is critical to understanding their biological functions. Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) combines parallel, high-throughput molecular analysis with location-specific information for the characterization of protein distributions directly from thin sections of intact biological tissue [1]. This technology enables discovery in biological systems and has unique advantages because it: (1) generates molecular-weight-specific data, (2) requires no target-specific reagents (such as antibodies), (3) can sample intact large proteins of 100 kDa, (4) is amenable to high-throughput protocols since it is capable of acquiring over 1,000 spectra per second with hundreds of proteins monitored in parallel in a single experiment, and (5) incorporates relatively simple and robust sample preparation. Moreover, MALDI IMS is complementary with other proteomic strategies like 2D gel electrophoresis and LC-MS/MS analysis since these require tissue homogenization that destroys spatial information and obscures small differences between regions of cells in tissue. MALDI uses a laser to desorb and ionize molecules in a sample that have been cocrystallized with a suitable matrix, typically a small aromatic molecule. Sample preparation is straightforward: a frozen section of tissue is cut 5- to 20-μm thick, thaw-mounted onto a MALDI target plate, matrix is applied directly to the tissue and allowed to air-dry. Matrix may be deposited using an automated matrix spotter (ca. 120-pL droplets) or by using an automated spray device or simple aerosol sprayer [2]. The method of matrix application and solvent system chosen may have an effect on the quality of MS spectra and these should be optimized for each project. Once the matrix is applied, a complete mass spectrum is acquired at specific (x,y) coordinates directly from the tissue, recording both the sampling location and ions detected. This is repeated in an ordered array across the substrate, where the sampling positions become `pixels' that are compiled to generate a picture or image for each molecule detected. Any given mass-to-charge (m/z) signal in the spectrum can be displayed with its relative intensity over the entire array, giving a density map of that compound in the array area. Hundreds of such pictures or images can be generated from a single acquisition experiment over such an array. The power of MALDI IMS technology lies in its capability to reliably combine protein data with specific cellular regions within the tissue, e.g., in the case shown in Fig. 1 for metastatic tumor cells and normal lymphocytes (laboratory, U. d. f. t. a. s, unpublished). The affected human lymph node shown is primarily composed of normal lymphocytes, with small pockets of metastatic melanoma tumor cells. Manual extraction of the tumor cells is difficult, and homogenization of the tissue would dilute the unique protein pool of the tumor cells. MALDI IMS images shown in Fig. 1 reveal several proteins observed at higher intensity in tumor cells (m/z 10,091, 10,627, and 11,643) and proteins with high intensity specific to lymphocytes (m/z 11,307, 13,375, and 15,327). The matrix array shown was robotically deposited at 150-μm spacing and in this case determines the lateral imaging resolution. Although spray deposition images are routinely acquired below 50-μm lateral resolution, robotic droplet deposition generally produces higher quality spectra. For reference, the average diameter of mammalian cells is ca. 10 μm. Fig. 1 MALDI IMS applied to a stage III metastatic melanoma invading the lymph node. a Optical hemotoxylin/eosin stain is shown with the tumor regions outlined in red. Matrix was robotically deposited at 150-μm spacing. b Ion density images of proteins ... Correlation of an observed m/z value in the MALDI spectra with a specific protein is done in the following manner. Typically, a small portion of the tissue is homogenized and proteins are isolated by HPLC and their molecular analytes are verified by MALDI MS. This isolate is further purified by gel electrophoresis, the gel band containing the protein of interest is removed, in-gel digested with trypsin, followed by LC-MS/MS analysis of the resulting peptides and comparison with a protein database. When possible the recovered theoretical molecular weight should match that determined experimentally, taking into account any loss of methionine, cystine disulfide bridges, acetylations, or other modifications from the protein database. Interest in MALDI imaging technology has grown among commercial vendors, who are now offering mass spectrometers with imaging capability, automated devices for matrix application, such as liquid jet dispensers and chemical printers, acoustic-driven spotters, and controlled spray deposition machines, and software/hardware solutions for MALDI image acquisition and data processing. Automated matrix application, either by picoliter droplet or spray coating, serves to reduce variations in matrix crystallization, deposition volumes, and drying times leading to a significant improvement in reproducibility, speed, and accuracy of matrix deposition [2]. The laser repetition rate is a crucial component for timely data acquisition, as many imaging experiments have 2,000 or more spots to be acquired. Commercial MALDI instruments are equipped with lasers having repetition rates of 200 Hz or more (i.e., 200 full spectra are acquired per second), with 1-kHz lasers soon to be available. For comparison, full data acquisition of an image with 2,000 spots or pixels and using 300 shots per spot would take nearly 1 h with a 200-Hz laser compared with a bit over 12 min with a 1-kHz laser.