From the journals: MCP

Young proteins are more likely to be degraded. Full automation speeds up sample preparation. Proteomics sheds light on cancer immune suppression. Read about recent papers on these topics published in the journal Molecular & Cellular Proteomics.

Young proteins are more likely to be degraded

The lifetime of proteins spans a wide range from minutes to months. Proteins involved in cell signaling typically turn over quickly to relay time-sensitive information. On the other hand, proteins critical to a cell’s basic functions usually have long lives to minimize metabolic costs. Scientists think that a protein’s lifetime could inform its function, suggesting that some cellular pathways may depend on protein age. Ubiquitination, or the addition of the small protein ubiquitin onto another protein, is an age–selective cellular process. It targets old or misfolded proteins for destruction by a protein complex known as the proteasome. A defective ubiquitin–proteasome system, or UPS, can lead to neurodegenerative disorders such as Alzheimer's disease.

In a recent paper published in the journal Molecular & Cellular Proteomics, Michael E. Meadow and colleagues at the University of Rochester, New York, described a novel approach, which they called “birthdating,” to profile the age of ubiquitinated proteins in human fibroblasts. Proteome-wide measurements of protein ages rely on a technique known as metabolic labeling, in which amino acid labels are added to cultured cells such that these labels are incorporated into newly synthesized proteins. In previous approaches, scientists introduced labels at the beginning of the experiment and collected samples at different time points. Birthdating reverses this process by introducing a new label at each successive time point and collecting a single sample for analysis at the end of the experiment. This process allows researchers to group proteins based on when they are synthesized.

The authors treated birthdated cells with a proteasome inhibitor, and then compared cellular proteomes in the presence and absence of the inhibitor. They observed significant changes, but only in newly synthesized proteins. The authors’ data suggests that the UPS selectively targets younger proteins for proteasomal degradation. They proposed that older proteins may be degraded via other pathways that don’t involve the proteasome.

Birthdating may allow scientists to partition the cellular proteome based on proteins’ ages as well as investigate the relationship between a protein’s age and its association with cellular pathways such as ubiquitination. These insights could lead to novel therapeutic targets for protein–misfolding diseases, such as Alzheimer's and Huntington’s disease.

Full automation speeds up sample preparation

In bottom-up proteomics, scientists enzymatically digest proteins into peptides before liquid chromatography-mass spectrometry, or LC-MS, analysis. Compared to intact proteins, peptides separate and fragment more predictably in LC-MS. Rapid technological advances have consistently made LC-MS runs faster and more sensitive. However, scientists are still hindered by time- and labor-intensive preparation steps for bottom-up proteomics.

Anders H. Kverneland, Florian Harking and a team of scientists in Denmark recently described a fully automated sample preparation workflow for bottom-up proteomics using a liquid-handling robot and the Evosep One platform for LC-MS. They published their results in the journal Molecular & Cellular Proteomics. Their approach eliminates manual sample handling, maximizing both throughput and reproducibility.

The authors demonstrated the clinical application of their workflow by analyzing the blood plasma proteomes of metastatic melanoma patients undergoing immunotherapy. By comparing the patients’ biomarker levels, they distinguished between patients who were responding to treatment and those who were not. They showed that this workflow can also be used for phosphoproteomics analysis, which can provide information on the activity of cell signaling pathways. Many diseases, such as cancer, are known to significantly alter the phosphoproteome.

Proteomics sheds light on cancer immunosuppression

The tumor microenvironment, or TME, is a complex ecosystem that surrounds a tumor. It consists of cancer-associated fibroblasts, blood vessels, extracellular matrix and immune cells. Cancer cells can secrete factors into the TME that suppress the activation of immune cells. This allows cancer cells to evade the immune surveillance, which can lead to tumor growth. In colorectal cancer, or CRC, immune cells are recruited to the mesenteric lymph nodes, or MLNs, that line the intestines. MLNs serve as the draining lymph nodes of CRC. Some studies have shown that immune cell activity is often suppressed in MLNs in CRC patients. However, scientists do not fully understand the mechanism behind this immunosuppression.

In a study published in the journal Molecular & Cellular Proteomics, a group in Taiwan, led by Jhih-Ci Yang, analyzed the TME and MLN proteome in a mouse model of CRC. The authors used a method known as library-based data-independent acquisition, or libDIA, in which they generated spectral libraries for six different types of immune cells. This significantly increased the sensitivity and coverage of immune cell proteins, which are usually hard to detect because they only make up a small population of the TME proteome. The authors showed that immune cells in MLNs downregulated signaling pathways involved in activation and differentiation. They hypothesized that these changes in the proteome likely contribute to the immunosuppression seen in MLNs.