Spatially-Resolved Proteomics: Rapid Quantitative Analysis of Laser Capture Microdissected Alveolar Tissue Samples

Abstract

Laser capture microdissection (LCM)-enabled region-specific tissue analyses are critical to better understand complex multicellular processes. However, current proteomics workflows entail several manual sample preparation steps and are challenged by the microscopic mass-limited samples generated by LCM, impacting measurement robustness, quantification and throughput. Here, we coupled LCM with a proteomics workflow that provides fully automated analysis of proteomes from microdissected tissues. Benchmarking against the current state-of-the-art in ultrasensitive global proteomics (FASP workflow), our approach demonstrated significant improvements in quantification (~2-fold lower variance) and throughput (>5 times faster). Using our approach we for the first time characterized, to a depth of >3,400 proteins, the ontogeny of protein changes during normal lung development in microdissected alveolar tissue containing only 4,000 cells. Our analysis revealed seven defined modules of coordinated transcription factor-signaling molecule expression patterns, suggesting a complex network of temporal regulatory control directs normal lung development with epigenetic regulation fine-tuning pre-natal developmental processes.

Advances in microscale separations towards nanoproteomics applications

“…The main bottleneck for the overall sensitivity lies in the sample losses and efficiency in the front-end sample processing. Recent developments in integrated online separation systems for microscale sample processing have demonstrated to be highly promising towards nanoproteomics analyses of small populations of cells [17,91,95,100]. While the current sensitivity of proteomics platforms still falls short of single cell analyses, continuous developments in the front-end integrated sample preparation techniques perhaps will be the most likely path in achieving such level of sensitivity.…”

“…4A) [17,91]. The SNaPP system created a simple, sensitive, robust, and reproducible nanoproteomics platform.…”

“…As a pilot study using this system, 348 proteins were identified from ~20 mouse blastocysts, equal to 100–200 ng samples [91]. More recently, >3400 proteins were identified by SNaPP from only 4000 lung cells isolated by LCM [17]. In another example, Tian et al .…”

“…Our group has recently coupled LCM with the SNaPP system to perform spatially resolved quantitative proteome profiling of microdissected lung alveolar tissue that was equivalent to only 4000 cells [17]. A depth of >3400 proteins was achieved on this platform with 2-fold lower quantification variance and 5-fold faster sample processing time when compared to offline sample processing.…”

“…1). Some of these sample types are readily produced by existing technologies such as fluorescence activated cell sorting(FACS) [15], laser capture dissection (LCM) [16,17], and exosome isolation techniques [18]. Moreover, single cell resolution genomics technologies, such as single-cell genomic sequencing [19] and single-cell transcriptomic profiling (RNA-Seq) [20,21], have been making tremendous impact in biological research.…”