To date, the effects of specific modification types and sites on protein lifetime have not been systematically illustrated. Here, we describe a proteomic method, DeltaSILAC, to quantitatively assess the impact of site-specific phosphorylation on the turnover of thousands of proteins in live cells. Based on the accurate and reproducible mass spectrometry-based method, a pulse labeling approach using stable isotope-labeled amino acids in cells (pSILAC), phosphoproteomics, and a unique peptide-level matching strategy, our DeltaSILAC profiling revealed a global, unexpected delaying effect of many phosphosites on protein turnover. We further found that phosphorylated sites accelerating protein turnover are functionally selected for cell fitness, enriched in Cyclin-dependent kinase substrates, and evolutionarily conserved, whereas the glutamic acids surrounding phosphosites significantly delay protein turnover. Our method represents a generalizable approach and provides a rich resource for prioritizing the effects of phosphorylation sites on protein lifetime in the context of cell signaling and disease biology.

• Klíčová slova
• DeltaSILAC, data-independent acquisition, mass spectrometry, phosphomodiform, phosphorylation, protein lifetime, protein turnover, proteomics, pulse SILAC,
• MeSH
• buněčný cyklus fyziologie   MeSH
• cyklin-dependentní kinasy genetika   metabolismus   MeSH
• fosfoproteiny chemie   metabolismus   MeSH
• fosforylace MeSH
• glutamáty metabolismus   MeSH
• hmotnostní spektrometrie metody   MeSH
• izotopové značení metody   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• peptidy metabolismus   MeSH
• peroxiredoxin VI chemie   metabolismus   MeSH
• proteolýza * MeSH
• proteom genetika   metabolismus   MeSH
• proteomika metody   MeSH
• sekvence aminokyselin MeSH
• sestřihové faktory chemie   metabolismus   MeSH
• signální transdukce genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• cyklin-dependentní kinasy MeSH
• fosfoproteiny MeSH
• glutamáty MeSH
• peptidy MeSH
• peroxiredoxin VI MeSH
• PRDX6 protein, human MeSH Prohlížeč
• proteom MeSH
• sestřihové faktory MeSH
• SF3B1 protein, human MeSH Prohlížeč

Profiling of biological relationships between different molecular layers dissects regulatory mechanisms that ultimately determine cellular function. To thoroughly assess the role of protein post-translational turnover, we devised a strategy combining pulse stable isotope-labeled amino acids in cells (pSILAC), data-independent acquisition mass spectrometry (DIA-MS), and a novel data analysis framework that resolves protein degradation rate on the level of mRNA alternative splicing isoforms and isoform groups. We demonstrated our approach by the genome-wide correlation analysis between mRNA amounts and protein degradation across different strains of HeLa cells that harbor a high grade of gene dosage variation. The dataset revealed that specific biological processes, cellular organelles, spatial compartments of organelles, and individual protein isoforms of the same genes could have distinctive degradation rate. The protein degradation diversity thus dissects the corresponding buffering or concerting protein turnover control across cancer cell lines. The data further indicate that specific mRNA splicing events such as intron retention significantly impact the protein abundance levels. Our findings support the tight association between transcriptome variability and proteostasis and provide a methodological foundation for studying functional protein degradation.

• Klíčová slova
• DIA mass spectrometry, alternative splicing, protein turnover, proteomics, pulsed SILAC,
• MeSH
• alternativní sestřih MeSH
• HeLa buňky MeSH
• hmotnostní spektrometrie MeSH
• izoformy RNA genetika   metabolismus   MeSH
• izotopové značení metody   MeSH
• lidé MeSH
• messenger RNA genetika   metabolismus   MeSH
• protein - isoformy analýza   metabolismus   MeSH
• proteiny analýza   metabolismus   MeSH
• proteolýza MeSH
• proteomika metody   MeSH
• průběh práce MeSH
• regulace genové exprese u nádorů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• izoformy RNA MeSH
• messenger RNA MeSH
• protein - isoformy MeSH
• proteiny MeSH

Due to the technical advances of mass spectrometers, particularly increased scanning speed and higher MS/MS resolution, the use of data-independent acquisition mass spectrometry (DIA-MS) became more popular, which enables high reproducibility in both proteomic identification and quantification. The current DIA-MS methods normally cover a wide mass range, with the aim to target and identify as many peptides and proteins as possible and therefore frequently generate MS/MS spectra of high complexity. In this report, we assessed the performance and benefits of using small windows with, e.g., 5-m/z width across the peptide elution time. We further devised a new DIA method named RTwinDIA that schedules the small isolation windows in different retention time blocks, taking advantage of the fact that larger peptides are normally eluting later in reversed phase chromatography. We assessed the direct proteomic identification by using shotgun database searching tools such as MaxQuant and pFind, and also Spectronaut with an external comprehensive spectral library of human proteins. We conclude that algorithms like pFind have potential in directly analyzing DIA data acquired with small windows, and that the instrumental time and DIA cycle time, if prioritized to be spent on small windows rather than on covering a broad mass range by large windows, will improve the direct proteome coverage for new biological samples and increase the quantitative precision. These results further provide perspectives for the future convergence between DDA and DIA on faster MS analyzers.

• Klíčová slova
• Data-independent acquisition, Isolation windows, Maxquant, Spectronaut, pFind,
• MeSH
• chromatografie s reverzní fází MeSH
• hmotnostní spektrometrie metody   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• peptidy analýza   MeSH
• proteiny analýza   MeSH
• proteomika metody   MeSH
• software MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• peptidy MeSH
• proteiny MeSH

Accurate classification of breast tumors is vital for patient management decisions and enables more precise cancer treatment. Here, we present a quantitative proteotyping approach based on sequential windowed acquisition of all theoretical fragment ion spectra (SWATH) mass spectrometry and establish key proteins for breast tumor classification. The study is based on 96 tissue samples representing five conventional breast cancer subtypes. SWATH proteotype patterns largely recapitulate these subtypes; however, they also reveal varying heterogeneity within the conventional subtypes, with triple negative tumors being the most heterogeneous. Proteins that contribute most strongly to the proteotype-based classification include INPP4B, CDK1, and ERBB2 and are associated with estrogen receptor (ER) status, tumor grade status, and HER2 status. Although these three key proteins exhibit high levels of correlation with transcript levels (R > 0.67), general correlation did not exceed R = 0.29, indicating the value of protein-level measurements of disease-regulated genes. Overall, this study highlights how cancer tissue proteotyping can lead to more accurate patient stratification.

• Klíčová slova
• SWATH-MS, breast cancer, data independent acquisition, proteomics, tissue, transcriptomics, tumor classification,
• MeSH
• fosfatasy genetika   metabolismus   MeSH
• lidé MeSH
• nádory prsu klasifikace   metabolismus   patologie   MeSH
• proteinkinasa CDC2 genetika   metabolismus   MeSH
• proteom analýza   metabolismus   MeSH
• proteomika metody   MeSH
• receptor erbB-2 genetika   metabolismus   MeSH
• receptory pro estrogeny metabolismus   MeSH
• rychlé screeningové testy MeSH
• tandemová hmotnostní spektrometrie metody   MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• CDK1 protein, human MeSH Prohlížeč
• ERBB2 protein, human MeSH Prohlížeč
• fosfatasy MeSH
• phosphatidylinositol-3,4-bisphosphate 4-phosphatase MeSH Prohlížeč
• proteinkinasa CDC2 MeSH
• proteom MeSH
• receptor erbB-2 MeSH
• receptory pro estrogeny MeSH

CRISPR-Cas gene editing holds substantial promise in many biomedical disciplines and basic research. Due to the important functional implications of non-histone chromosomal protein HMG-14 (HMGN1) in regulating chromatin structure and tumor immunity, gene knockout of HMGN1 is performed by CRISPR in cancer cells and the following proteomic regulation events are studied. In particular, DIA mass spectrometry (DIA-MS) is utilized, and more than 6200 proteins (protein- FDR 1%) and more than 82 000 peptide precursors are reproducibly measured in the single MS shots of 2 h. HMGN1 protein deletion is confidently verified by DIA-MS in all of the clone- and dish- replicates following CRISPR. Statistical analysis reveals 147 proteins change their expressions significantly after HMGN1 knockout. Functional annotation and enrichment analysis indicate the deletion of HMGN1 induces histone inactivation, various stress pathways, remodeling of extracellular proteomes, cell proliferation, as well as immune regulation processes such as complement and coagulation cascade and interferon alpha/ gamma response in cancer cells. These results shed new lights on the cellular functions of HMGN1. It is suggested that DIA-MS can be reliably used as a rapid, robust, and cost-effective proteomic-screening tool to assess the outcome of the CRISPR experiments.

• Klíčová slova
• CRISPR-Cas9, HMGN1, clone effect, data independent acquisition, gene deletion, protein function,
• MeSH
• chromatin fyziologie   MeSH
• CRISPR-Cas systémy MeSH
• delece genu * MeSH
• editace genu metody   MeSH
• HeLa buňky MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• proliferace buněk genetika   MeSH
• protein HMGN1 genetika   MeSH
• proteomika metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chromatin MeSH
• protein HMGN1 MeSH

Targeted mass spectrometry-based proteomics approaches enable the simultaneous and reproducible quantification of multiple protein analytes across numerous conditions in biology and clinical studies. These approaches involve e.g. selected reaction monitoring (SRM) typically conducted on a triple quadrupole mass spectrometer, its high-resolution variant named pseudo-SRM (p-SRM), carried out in a quadrupole coupled with an TOF analyzer (qTOF), and "sequential window acquisition of all theoretical spectra" (SWATH). Here we compared these methods in terms of signal-to-noise ratio (S/N), coefficient of variance (CV), fold change (FC), limit of detection and quantitation (LOD, LOQ). We have shown the highest S/N for p-SRM mode, followed by SRM and SWATH, demonstrating a trade-off between sensitivity and level of multiplexing for SRM, p-SRM, and SWATH. SRM was more sensitive than p-SRM based on determining their LOD and LOQ. Although SWATH has the worst S/N, it enables peptide multiplexing with post-acquisition definition of the targets, leading to better proteome coverage. FC between breast tumors of different clinical-pathological characteristics were highly correlated (R2 >0.97) across three methods and consistent with the previous study on 96 tumor tissues. Our technical note presented here, therefore, confirmed that outputs of all the three methods were biologically relevant and highly applicable to cancer research.

• Klíčová slova
• Cancer, MRM/SRM, SWATH, Tissue, p-SRM,
• MeSH
• hmotnostní spektrometrie přístrojové vybavení   metody   MeSH
• lidé MeSH
• limita detekce MeSH
• nádory chemie   metabolismus   MeSH
• poměr signál - šum MeSH
• proteiny analýza   MeSH
• proteomika metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• proteiny MeSH

Non-invasive detection of colorectal cancer with blood-based markers is a critical clinical need. Here we describe a phased mass spectrometry-based approach for the discovery, screening, and validation of circulating protein biomarkers with diagnostic value. Initially, we profiled human primary tumor tissue epithelia and characterized about 300 secreted and cell surface candidate glycoproteins. These candidates were then screened in patient systemic circulation to identify detectable candidates in blood plasma. An 88-plex targeting method was established to systematically monitor these proteins in two large and independent cohorts of plasma samples, which generated quantitative clinical datasets at an unprecedented scale. The data were deployed to develop and evaluate a five-protein biomarker signature for colorectal cancer detection.

• Klíčová slova
• colorectal cancer, diagnostic protein biomarker, discovery‐driven and targeted proteomics,
• MeSH
• hmotnostní spektrometrie metody   MeSH
• klinické laboratorní techniky metody   MeSH
• kolorektální nádory diagnóza   MeSH
• krevní plazma chemie   MeSH
• lidé MeSH
• nádorové biomarkery krev   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH
• Názvy látek
• nádorové biomarkery MeSH