Optimized Data-Independent Acquisition Approach for Proteomic Analysis at Single-Cell Level

Background: Single-cell proteomic analysis provides valuable insights into cellular heterogeneity allowing the characterization of the cellular microenvironment which is difficult to accomplish in bulk proteomic analysis. Currently, single-cell proteomic studies utilize data-dependent acquisition (DDA) mass spectrometry (MS) coupled with a TMT labelled carrier channel. Due to the extremely imbalanced MS signals among the carrier channel and other TMT reporter ions, the quantification is compromised. Thus, data-independent acquisition (DIA)-MS should be considered as an alternative approach towards single-cell proteomic study since it generates reproducible quantitative data. However, there are limited reports on the optimal workflow for DIA-MS-based single-cell analysis. Methods: We report an optimized DIA workflow for single-cell proteomics using Orbitrap Lumos Tribrid instrument. We utilized a breast cancer cell line (MDA-MB-231) and induced drug resistant polyaneuploid cancer cells (PACCs) to evaluate our established workflow. Results: We found that a short LC gradient was preferable for peptides extracted from single cell level with less than 2 ng sample amount. The total number of co-searching peptide precursors was also critical for protein and peptide identifications at nano- and sub-nano-gram levels. Post-translationally modified peptides could be identified from a nano-gram level of peptides. Using the optimized workflow, up to 1,500 protein groups were identified from a single PACC corresponding to 0.2 ng of peptides. Furthermore, about 200 peptides with phosphorylation, acetylation, and ubiquitination were identified from global DIA analysis of 100 cisplatin resistant PACCs (20 ng). Finally, we used this optimized DIA approach to compare the whole proteome of MDA-MB-231 parental cells and induced PACCs at a single-cell level. We found the single-cell level comparison could reflect real protein expression changes and identify the protein copy number. Conclusions: Our results demonstrate that the optimized DIA pipeline can serve as a reliable quantitative tool for single-cell as well as sub-nano-gram proteomic analysis.

Structural basis for the peptidoglycan editing activity of YfiH

Bacterial cells are encased in peptidoglycan (PG), a polymer of disaccharide N-acetyl-glucosamine (GlcNAc) and N-acetyl-muramic acid (MurNAc) cross-linked by peptide stems. PG is synthesized in the cytoplasm as UDP-MurNAc-peptide precursors, of which the amino-acid composition of the peptide is unique, with L-Ala added at the first position in most bacteria but L-Ser or Gly in some bacteria. YfiH is a PG-editing factor whose absence causes misincorporation of L-Ser instead of L-Ala into peptide stems; but its mechanistic function is unknown. Here we report the crystal structures of substrate-bound and product-bound YfiH, showing that YfiH is a cytoplasmic amidase that controls the incorporation of the correct amino acid to the nucleotide precursors by preferentially cleaving the nucleotide precursor byproduct UDP-MurNAc-L-Ser. This work reveals an editing mechanism in the cytoplasmic steps of peptidoglycan biosynthesis.

Optimized Data-Independent Acquisition Approach for Proteomic Analysis at Single-Cell Level

Single-cell proteomic analysis provides valuable insights into cellular heterogeneity allowing the characterization of the cellular microenvironment which is difficult to accomplish in bulk proteomic analysis. Currently, single-cell proteomic studies utilize data-dependent acquisition (DDA) mass spectrometry (MS) coupled with a TMT labelled carrier channel. Due to the extremely imbalanced MS signals among the carrier channel and other TMT reporter ions, the quantification is compromised. Thus, data-independent acquisition (DIA)-MS should be considered as an alternative approach towards single-cell proteomic study since it generates reproducible quantitative data. However, there are limited reports on the optimal workflow for DIA-MS-based single-cell analysis. Herein, we report an optimized DIA workflow for single-cell proteomics using Orbitrap Lumos Tribrid instrument. We utilized a breast cancer cell line (MDA-MB-231) and induced drug resistant polyaneuploid cancer cells (PACCs) to evaluate our established workflow. We found that a short LC gradient was preferable for peptides extracted from single cell level with less than 2 ng sample amount. The total number of co-searching peptide precursors was also critical for protein and peptide identifications at nano- and sub-nano-gram levels. Post-translationally modified peptides could be identified from a nano-gram level of peptides. Using the optimized workflow, up to 1,500 protein groups were identified from a single PACC corresponding to 0.2 ng of peptides. Furthermore, about 200 peptides with phosphorylation, acetylation, and ubiquitination were identified from global DIA analysis of 100 cisplatin resistant PACCs (20 ng). Finally, we used this optimized DIA approach to compare the whole proteome of MDA-MB-231 parental cells and induced PACCs at a single-cell level. We found the single-cell level comparison could reflect real protein expression changes and identify the protein copy number. Our results demonstrate that the optimized DIA pipeline can serve as a reliable quantitative tool for single-cell as well as sub-nano-gram proteomic analysis.

Evolution and Potential Function in Molluscs of Neuropeptide and Receptor Homologues of the Insect Allatostatins

The allatostatins (ASTs), AST-A, AST-B and AST-C, have mainly been investigated in insects. They are a large group of small pleotropic alloregulatory neuropeptides that are unrelated in sequence and activate receptors of the rhodopsin G-protein coupled receptor family (GPCRs). The characteristics and functions of the homologue systems in the molluscs (Buccalin, MIP and AST-C-like), the second most diverse group of protostomes after the arthropods, and of high interest for evolutionary studies due to their less rearranged genomes remains to be explored. In the present study their evolution is deciphered in molluscs and putative functions assigned in bivalves through meta-analysis of transcriptomes and experiments. Homologues of the three arthropod AST-type peptide precursors were identified in molluscs and produce a larger number of mature peptides than in insects. The number of putative receptors were also distinct across mollusc species due to lineage and species-specific duplications. Our evolutionary analysis of the receptors identified for the first time in a mollusc, the cephalopod, GALR-like genes, which challenges the accepted paradigm that AST-AR/buccalin-Rs are the orthologues of vertebrate GALRs in protostomes. Tissue transcriptomes revealed the peptides, and their putative receptors have a widespread distribution in bivalves and in the bivalve Mytilus galloprovincialis, elements of the three peptide-receptor systems are highly abundant in the mantle an innate immune barrier tissue. Exposure of M. galloprovincialis to lipopolysaccharide or a marine pathogenic bacterium, Vibrio harveyi, provoked significant modifications in the expression of genes of the peptide precursor and receptors of the AST-C-like system in the mantle suggesting involvement in the immune response. Overall, our study reveals that homologues of the arthropod AST-systems in molluscs are potentially more complex due to the greater number of putative mature peptides and receptor genes. In bivalves they have a broad and varying tissue distribution and abundance, and the elements of the AST-C-like family may have a putative function in the immune response.

Thimet Oligopeptidase—A Classical Enzyme with New Function and New Form

Peptidases generate bioactive peptides that can regulate cell signaling and mediate intercellular communication. While the processing of peptide precursors is initiated intracellularly, some modifications by peptidases may be conducted extracellularly. Thimet oligopeptidase (TOP) is a peptidase that processes neuroendocrine peptides with roles in mood, metabolism, and immune responses, among other functions. TOP also hydrolyzes angiotensin I to angiotensin 1–7, which may be involved in the pathophysiology of COVID-19 infection. Although TOP is primarily cytosolic, it can also be associated with the cell plasma membrane or secreted to the extracellular space. Recent work indicates that membrane-associated TOP can be released with extracellular vesicles (EVs) to the extracellular space. Here we briefly summarize the enzyme’s classical function in extracellular processing of neuroendocrine peptides, as well as its more recently understood role in intracellular processing of various peptides that impact human diseases. Finally, we discuss new findings of EV-associated TOP in the extracellular space.

Optimized Data-Independent Acquisition Approach for Proteomic Analysis at Single-Cell Level

Single-cell proteomic analysis provides valuable insights into cellular heterogeneity allowing the characterization of the cellular microenvironment which is difficult to accomplish in bulk proteomic analysis. Currently, single-cell proteomic studies utilize data-dependent acquisition (DDA) mass spectrometry (MS) coupled with a TMT labelled carrier channel. Due to the extremely imbalanced MS signals among the carrier channel and other TMT reporter ions, the quantification is compromised. Thus, data-independent acquisition (DIA)-MS should be considered as an alternative approach towards single-cell proteomic study since it generates reproducible quantitative data. However, there are limited reports on the optimal workflow for DIA-MS-based single-cell analysis. Herein, we report an optimized DIA workflow for single-cell proteomics using Orbitrap Lumos Tribrid instrument. We utilized a breast cancer cell line (MDA-MB-231) and induced drug resistant polyaneuploid cancer cells (PACCs) to evaluate our established workflow. We found that a short LC gradient was preferable for peptides extracted from single cell level with less than 2 ng sample amount. The total number of co-searching peptide precursors was also critical for protein and peptide identifications at nano- and sub-nano-gram levels. Post-translationally modified peptides could be identified from a nano-gram level of peptides. Using the optimized workflow, up to 1,500 protein groups were identified from a single PACC corresponding to 0.2 ng of peptides. Furthermore, about 200 peptides with phosphorylation, acetylation, and ubiquitination were identified from global DIA analysis of 100 cisplatin resistant PACCs (20 ng). Finally, we used this optimized DIA approach to compare the whole proteome of MDA-MB-231 parental cells and induced PACCs at a single-cell level. We found the single-cell level comparison could reflect real protein expression changes and identify the protein copy number. Our results demonstrates that the optimized DIA pipeline can serve as a reliable quantitative tool for single-cell as well as sub-nano-gram proteomic analysis.

Real-Time Search Assisted Acquisition on a Tribrid Mass Spectrometer Improves Coverage in Multiplexed Single-Cell Proteomics

In the young field of single-cell proteomics (scMS), there is a great need for improved global proteome characterization, both in terms of proteins quantified per cell and quantitative performance thereof. The recently introduced real-time search (RTS) on the Orbitrap Eclipse Tribrid mass spectrometer in combination with SPS-MS3 acquisition has been shown to be beneficial for the measurement of samples that are multiplexed using isobaric tags. Multiplexed single-cell proteomics requires high ion injection times and high-resolution spectra to quantify the single-cell signal, however the booster channel facilitates peptide identification and thus offers the opportunity for fast on the fly peptide identification before committing to the time intensive quantification scan. Here, we compared classical MS2 acquisition against RTS-SPS-MS3, both using the Orbitrap Eclipse Tribrid MS with the FAIMS Pro ion mobility interface and we present a new acquisition strategy that makes use of fast real-time searched linear ion trap scans to preselect MS1 peptide precursors for quantitative MS2 Orbitrap acquisition. Here we show that this strategy, termed RETICLE (RTS Enhanced Quant of Single Cell Spectra) outperforms MS2 and RTS-SPS-MS3 in multiplexed scMS applications and enables the quantification of over 1000 proteins per cell at a MS2 injection time of 750ms using a 2h gradient, resulting in improved quality of scMS datasets.

The ERAP1 active site cannot productively access the N-terminus of antigenic peptide precursors stably bound onto MHC class I

Processing of N-terminally elongated antigenic peptide precursors by Endoplasmic Reticulum Aminopeptidase 1 (ERAP1) is a key step in antigen presentation and the adaptive immune response. Although ERAP1 can efficiently process long peptides in solution, it has been proposed that it can also process peptides bound onto Major Histocompatibility Complex I molecules (MHCI). In a previous study, we suggested that the occasionally observed “ontο MHCI” trimming by ERAP1 is likely due to fast peptide dissociation followed by solution trimming, rather than direct action of ERAP1 onto the MHCI complex. However, other groups have proposed that ERAP1 can trim peptides covalently bound onto MHCI, which would preclude peptide dissociation. To explore this interaction, we constructed disulfide-linked MHCI-peptide complexes using HLA-B*08 and a 12mer kinetically labile peptide, or a 16mer carrying a phosphinic transition-state analogue N-terminus with high-affinity for ERAP1. Kinetic and biochemical analyses suggested that while both peptides could access the ERAP1 active site when free in solution, they were unable to do so when tethered in the MHCI binding groove. Our results suggest that MHCI binding protects, rather than promotes, antigenic peptide precursor trimming by ERAP1 and thus solution trimming is the more likely model of antigenic peptide processing.

Cyclic Dipeptides Formation From Linear Dipeptides Under Potentially Prebiotic Earth Conditions

Cyclic dipeptides (DKPs) are peptide precursors and chiral catalysts in the prebiotic process. This study reports proline-containing DKPs that were spontaneously obtained from linear dipeptides under an aqueous solution. Significantly, the yields of DKPs were affected by the sequence of linear dipeptides and whether the reaction contains trimetaphosphate. These findings provide the possibility that DKPs might play a key role in the origin of life.