Sensitive detection of low-abundance proteins in complex biological samples has typically been achieved by immunoassays that use antibodies specific to target proteins; however, de novo development of antibodies is associated with high costs, long development lead times, and high failure rates. To address these challenges, we developed an antibody-free strategy that involves PRISM (high-pressure, high-resolution separations coupled with intelligent selection and multiplexing) for sensitive selected reaction monitoring (SRM)-based targeted protein quantification. The strategy capitalizes on high-resolution reversed-phase liquid chromatographic separations for analyte enrichment, intelligent selection of target fractions via on-line SRM monitoring of internal standards, and fraction multiplexing before nano-liquid chromatography-SRM quantification. Application of this strategy to human plasma/serum demonstrated accurate and reproducible quantification of proteins at concentrations in the 50-100 pg/mL range, which represents a major advance in the sensitivity of targeted protein quantification without the need for specific-affinity reagents. Application to a set of clinical serum samples illustrated an excellent correlation between the results obtained from the PRISM-SRM assay and those from clinical immunoassay for the prostate-specific antigen level.biomarker verification | systems biology | targeted proteomics S elected reaction monitoring (SRM), also known as multiple reaction monitoring (MRM), has recently emerged as a promising technology (1-16) for high-throughput mass spectrometry (MS)-based quantification of targeted proteins in biological and clinical specimens (e.g., tumor tissues). SRM has demonstrated relatively good selectivity, reproducibility (or precision), and sensitivity for a range of multiplexed protein assays (2, 4-8, 11, 17, 18) and has potential for quantifying protein isoforms (19) and posttranslational modifications (PTMs) (3,(20)(21)(22) for which good quality antibodies often do not exist. Nevertheless, a major limitation of current SRM technology is insufficient sensitivity for detecting low-abundance proteins present at sub-nanogram per milliliter levels in human blood plasma or serum and extremely low-abundance proteins in cells or tissues. Without sample prefractionation, liquid chromatography (LC)-SRM measurements have been limited to only moderately abundant proteins in human plasma present in the low microgram per milliliter range (2,6,8).More recently, the combination of immunoaffinity depletion and fractionation by strong cation exchange (SCX) chromatography (4, 23), along with advances in MS sensitivity (24), has extended SRM quantification of plasma proteins to low nanogram per milliliter levels (4, 23). SISCAPA (stable isotope standards and capture by anti-peptide antibodies) coupled with SRM demonstrated quantification of target proteins in the same range, using as little as 10 μL of human plasma (8,(25)(26)(27). SISCAPA assays have some distinct advantages over conventiona...

The mitochondria-associated membrane (MAM) has emerged as a cellular signaling hub regulating various cellular processes. However, its molecular components remain unclear owing to lack of reliable methods to purify the intact MAM proteome in a physiological context. Here, we introduce Contact-ID, a split-pair system of BioID with strong activity, for identification of the MAM proteome in live cells. Contact-ID specifically labeled proteins proximal to the contact sites of the endoplasmic reticulum (ER) and mitochondria, and thereby identified 115 MAM-specific proteins. The identified MAM proteins were largely annotated with the outer mitochondrial membrane (OMM) and ER membrane proteins with MAM-related functions: e.g., FKBP8, an OMM protein, facilitated MAM formation and local calcium transport at the MAM. Furthermore, the definitive identification of biotinylation sites revealed membrane topologies of 85 integral membrane proteins. Contact-ID revealed regulatory proteins for MAM formation and could be reliably utilized to profile the proteome at any organelle–membrane contact sites in live cells.

The inner mitochondrial membrane (IMM) proteome plays a central role in maintaining mitochondrial physiology and cellular metabolism. Various important biochemical reactions such as oxidative phosphorylation, metabolite production, and mitochondrial biogenesis are conducted by the IMM proteome, and mitochondria-targeted therapeutics have been developed for IMM proteins, which is deeply related for various human metabolic diseases including cancer and neurodegenerative diseases. However, the membrane topology of the IMM proteome remains largely unclear because of the lack of methods to evaluate it in live cells in a high-throughput manner. In this article, we reveal the in vivo topological direction of 135 IMM proteins, using an in situ-generated radical probe with genetically targeted peroxidase (APEX). Owing to the short lifetime of phenoxyl radicals generated in situ by submitochondrial targeted APEX and the impermeability of the IMM to small molecules, the solvent-exposed tyrosine residues of both the matrix and intermembrane space (IMS) sides of IMM proteins were exclusively labeled with the radical probe in live cells by Matrix-APEX and IMS-APEX, respectively and identified by mass spectrometry. From this analysis, we confirmed 58 IMM protein topologies and we could determine the topological direction of 77 IMM proteins whose topology at the IMM has not been fully characterized. We also found several IMM proteins (e.g., LETM1 and OXA1) whose topological information should be revised on the basis of our results. Overall, our identification of structural information on the mitochondrial inner-membrane proteome can provide valuable insights for the architecture and connectome of the IMM proteome in live cells.