Mass spectrometry (MS)-based proteomics typically employs multistep sample-preparation workflows that are subject to sample contamination and loss. We report an in-StageTip method for performing sample processing, from cell lysis through elution of purified peptides, in a single, enclosed volume. This robust and scalable method largely eliminates contamination or loss. Peptides can be eluted in several fractions or in one step for single-run proteome analysis. In one day, we obtained the largest proteome coverage to date for budding and fission yeast, and found that protein copy numbers in these cells were highly correlated (R(2) = 0.78). Applying the in-StageTip method to quadruplicate measurements of a human cell line, we obtained copy-number estimates for 9,667 human proteins and observed excellent quantitative reproducibility between replicates (R(2) = 0.97). The in-StageTip method is straightforward and generally applicable in biological or clinical applications.

Circular RNAs (circRNAs) are broadly expressed in eukaryotic cells, but their molecular mechanism in human disease remains obscure. Here we show that circular antisense non-coding RNA in the INK4 locus (circANRIL), which is transcribed at a locus of atherosclerotic cardiovascular disease on chromosome 9p21, confers atheroprotection by controlling ribosomal RNA (rRNA) maturation and modulating pathways of atherogenesis. CircANRIL binds to pescadillo homologue 1 (PES1), an essential 60S-preribosomal assembly factor, thereby impairing exonuclease-mediated pre-rRNA processing and ribosome biogenesis in vascular smooth muscle cells and macrophages. As a consequence, circANRIL induces nucleolar stress and p53 activation, resulting in the induction of apoptosis and inhibition of proliferation, which are key cell functions in atherosclerosis. Collectively, these findings identify circANRIL as a prototype of a circRNA regulating ribosome biogenesis and conferring atheroprotection, thereby showing that circularization of long non-coding RNAs may alter RNA function and protect from human disease.

Proteins in the circulatory system mirror an individual's physiology. In daily clinical practice, protein levels are generally determined using single-protein immunoassays. High-throughput, quantitative analysis using mass-spectrometry-based proteomics of blood, plasma, and serum would be advantageous but is challenging because of the high dynamic range of protein abundances. Here, we introduce a rapid and robust "plasma proteome profiling" pipeline. This single-run shotgun proteomic workflow does not require protein depletion and enables quantitative analysis of hundreds of plasma proteomes from 1 μl single finger pricks with 20 min gradients. The apolipoprotein family, inflammatory markers such as C-reactive protein, gender-related proteins, and >40 FDA-approved biomarkers are reproducibly quantified (CV <20% with label-free quantification). Furthermore, we functionally interpret a 1,000-protein, quantitative plasma proteome obtained by simple peptide pre-fractionation. Plasma proteome profiling delivers an informative portrait of a person's health state, and we envision its large-scale use in biomedicine.

Yeast remains an important model for systems biology and for evaluating proteomics strategies. In-depth shotgun proteomics studies have reached nearly comprehensive coverage, and rapid, targeted approaches have been developed for this organism. Recently, we demonstrated that single LC-MS/MS analysis using long columns and gradients coupled to a linear ion trap Orbitrap instrument had an unexpectedly large dynamic range of protein identification (Thakur, S. S., Geiger, T., Chatterjee, B., Bandilla, P., Frohlich, F., Cox, J., and Mann, M.

High-resolution mass spectrometry (MS)-based proteomics has progressed tremendously over the years. For model organisms like yeast, we can now quantify complete proteomes in just a few hours. Developments discussed in this Perspective will soon enable complete proteome analysis of mammalian cells, as well, with profound impact on biology and biomedicine.