One-Shot Single-Cell Proteome and Metabolome Analysis Strategy for the Same Single Cell.

Characterizing the profiles of proteome and metabolome at the single-cell level is of great significance in single-cell multiomic studies. Herein, we proposed a novel strategy called one-shot single-cell proteome and metabolome analysis (scPMA) to acquire the proteome and metabolome information in a single-cell individual in one injection of LC-MS/MS analysis. Based on the scPMA strategy, a total workflow was developed to achieve the single-cell capture, nanoliter-scale sample pretreatment, one-shot LC injection and separation of the enzyme-digested peptides and metabolites, and dual-zone MS/MS detection for proteome and metabolome profiling. Benefiting from the scPMA strategy, we realized dual-omic analysis of single tumor cells, including A549, HeLa, and HepG2 cells with 816, 578, and 293 protein groups and 72, 91, and 148 metabolites quantified on average. A single-cell perspective experiment for investigating the doxorubicin-induced antitumor effects in both the proteome and metabolome aspects was also performed.

ß-Receptor blocker enhances the anabolic effect of PTH after osteoporotic fracture.

The autonomic nervous system plays a crucial role in regulating bone metabolism, with sympathetic activation stimulating bone resorption and inhibiting bone formation. We found that fractures lead to increased sympathetic tone, enhanced osteoclast resorption, decreased osteoblast formation, and thus hastened systemic bone loss in ovariectomized (OVX) mice. However, the combined administration of parathyroid hormone (PTH) and the ß-receptor blocker propranolol dramatically promoted systemic bone formation and osteoporotic fracture healing in OVX mice. The effect of this treatment is superior to that of treatment with PTH or propranolol alone. In vitro, the sympathetic neurotransmitter norepinephrine (NE) suppressed PTH-induced osteoblast differentiation and mineralization, which was rescued by propranolol. Moreover, NE decreased the PTH-induced expression of Runx2 but enhanced the expression of Rankl and the effect of PTH-stimulated osteoblasts on osteoclastic differentiation, whereas these effects were reversed by propranolol. Furthermore, PTH increased the expression of the circadian clock gene Bmal1, which was inhibited by NE-ßAR signaling. Bmal1 knockdown blocked the rescue effect of propranolol on the NE-induced decrease in PTH-stimulated osteoblast differentiation. Taken together, these results suggest that propranolol enhances the anabolic effect of PTH in preventing systemic bone loss following osteoporotic fracture by blocking the negative effects of sympathetic signaling on PTH anabolism.

Pick-up single-cell proteomic analysis for quantifying up to 3000 proteins in a Mammalian cell.

The shotgun proteomic analysis is currently the most promising single-cell protein sequencing technology, however its identification level of ~1000 proteins per cell is still insufficient for practical applications. Here, we develop a pick-up single-cell proteomic analysis (PiSPA) workflow to achieve a deep identification capable of quantifying up to 3000 protein groups in a mammalian cell using the label-free quantitative method. The PiSPA workflow is specially established for single-cell samples mainly based on a nanoliter-scale microfluidic liquid handling robot, capable of achieving single-cell capture, pretreatment and injection under the pick-up operation strategy. Using this customized workflow with remarkable improvement in protein identification, 2449-3500, 2278-3257 and 1621-2904 protein groups are quantified in single A549 cells (n = 37), HeLa cells (n = 44) and U2OS cells (n = 27) under the DIA (MBR) mode, respectively. Benefiting from the flexible cell picking-up ability, we study HeLa cell migration at the single cell proteome level, demonstrating the potential in practical biological research from single-cell insight.

Investigating the ibrutinib resistance mechanism of L528W mutation on Bruton's tyrosine kinase via molecular dynamics simulations.

Drug resistance to Bruton's Tyrosine Kinase (BTK) inhibitors presents a challenge in treating B-cell malignancies, and the mechanism behind drug resistance remains unclear. In this study, we focused on the BTK L528W mutation and investigated the underlying mechanisms of resistance to ibrutinib (including prototype and its active metabolite from, PCI-45227) using a combination of bioinformatics analysis, and molecular dynamics (MD) simulations. Protein stability of wild type (WT) BTK and L528W mutant was predicted using DUET, PoPMuSiC, and I-Mutant2.0. We performed MD simulations of six systems, apo-WT, metabolite-WT, prototype-WT and their mutants, to analyze the significant conformational and BTK-inhibitor binding affinity changes induced by the L528W mutation. Results show that the L528W mutation reduces the conformational stability of BTK compared to the WT. Principal component analysis (PCA) based free energy landscape (FEL) analysis shows that the L528W mutant ensemble tends to form more conformation clusters and exhibit higher levels of local minima than the WT counterpart. The interaction analysis reveal that the L528W mutation disrupts the strong hydrogen bond between Cys481 and inhibitors and reduces the number of hydrogen bonds between inhibitors and BTK in the L528W mutant complex structures compared to the WT. Porcupine plot analysis in association with cross-correlation analysis show the high-intensity flexible motion exhibited by the P-loop region. MM/GBSA calculations show that the L528W mutation in metabolite-BTK and prototype-BTK complexes increases binding free energy compared to the WT, with a reduction in binding affinity confirmed by per-residue energy decomposition. Specifically, the binding free energy increases from -57.86 kcal/mol to -48.26 kcal/mol for the metabolite-BTK complex and from -62.04 kcal/mol to -50.55 kcal/mol for the prototype-BTK complex. Overall, our study finds that the L528W mutation reduces BTK stability, decreases binding affinity, and leads to drug resistance and potential disease recurrence.

18 months follow-up of deep molecular response 4.5 (MR4.5) with nilotinib in patients with newly diagnosed chronic-phase chronic myeloid leukemia: a prospective, multi-center study in China.

Introduction: Early stable deep molecular response (DMR) to nilotinib is associated with goal of treatment-free remission (TFR) in patients with chronic-phase chronic myeloid leukemia (CML-CP). It is important to early distinguish between patients who can achieve a DMR and those who are fit for TFR. Methods: We performed a multicenter study to explore the early cumulative MR4.5 rate at 18 months with nilotinib in patients with newly diagnosed CML-CP (ND-CML-CP) in China. Of the 29 institutes, 106 patients with ND-CML-CP received nilotinib (300 mg BID). Results and discussion: The cumulative MR4.5 rate of nilotinib treatment at 18 months was 69.8% (74/106). The cumulative MMR and MR4.0 rates for nilotinib at 18 months were 94.3% (100/106) and 84.9% (90/106), respectively. Patients with an ultra-early molecular response (u-EMR) at 6 weeks were not significantly different in obtaining DMR or MMR by 24 months compared with those without u-EMR (p = 0.7584 and p = 0.9543, respectively). Our study demonstrated that nilotinib treatment in patients with ND-CML-CP contributed to obtain high early MR4.5.

Simultaneous deep transcriptome and proteome profiling in a single mouse oocyte.

Although single-cell multi-omics technologies are undergoing rapid development, simultaneous transcriptome and proteome analysis of a single-cell individual still faces great challenges. Here, we developed a single-cell simultaneous transcriptome and proteome (scSTAP) analysis platform based on microfluidics, high-throughput sequencing, and mass spectrometry technology to achieve deep and joint quantitative analysis of transcriptome and proteome at the single-cell level, providing an important resource for understanding the relationship between transcription and translation in cells. This platform was applied to analyze single mouse oocytes at different meiotic maturation stages, reaching an average quantification depth of 19,948 genes and 2,663 protein groups in single mouse oocytes. In particular, we analyzed the correlation of individual RNA and protein pairs, as well as the meiosis regulatory network with unprecedented depth, and identified 30 transcript-protein pairs as specific oocyte maturational signatures, which could be productive for exploring transcriptional and translational regulatory features during oocyte meiosis.

CD4 T cells in chronic myeloid leukemia present MHC class II-dependent and IFN-γ-dependent cytotoxic capacity.

At present, many therapeutic schemes have been used to improve the prognosis of patients with chronic myeloid leukemia (CML), but response remains poor in a small group of patients. CD4 T cell-mediated cytotoxicity has been found in various autoimmune diseases. This study analyzed the characteristics of CD4 T cell mediated cytotoxicity in CML patients and healthy people. The cytotoxicity of CD4 T cells was tested in using two CML cell lines, including the MHC class II-deficient K562 cells and the MHC class II-expressing KU812 cells. CD4 T cell-mediated lysis was minimal in K562 cells but was much higher in KU812 cells. In CML patients, the level of CD4 T cell-mediated lysis was limited to a certain level. Interestingly, pre-treating KU812 cells with IFN-γ could significantly elevate the expression of MHC class II and elevate the level of CD4 T cell-mediated lysis. Overall, these data indicated CD4 T cells could become a potential candidate for cytotoxic elimination of CML cells.

Evaluation of physician guideline adherence and areas for improvement in managing patients with chronic myeloid leukemia: a cross-sectional survey.

The experience of a physician at a clinical center is among the critical factors in managing chronic myeloid leukemia (CML) during its treatment with tyrosine kinase inhibitors (TKIs). The authors conducted a cross-sectional questionnaire to investigate barriers to physician use of published evidence-based guidelines in CML management in a real-world setting. Among the participating physicians (N = 407), 99.8% of physicians reported that CML guidelines were useful; however, only 62.9% of physicians reported that they follow guidelines in real-time. Although 90.7% of physicians prefer second-generation TKIs as the first-line treatment, imatinib (88.2%) remains the most widely administered TKI in the first-line setting. Only 50.6% of physicians switched the treatment when patients failed to achieve early molecular response (at 3 months), whereas 70.3% of physicians switched the treatment when patients' response to TKI was inadequate at 6 months and/or 12 months. Moreover, only 43.5% of physicians considered treatment-free remission (TFR) as one of the top 3 goals for their patients. The major concern to obtain TFR was patients' adherence. This study demonstrated that CML management was generally in line with the current guidelines, but some of the details at the point of care are needed to be improved in CML.

Orelabrutinib and venetoclax synergistically induce cell death in double-hit lymphoma by interfering with the crosstalk between the PI3K/AKT and p38/MAPK signaling.

PURPOSE: Double-hit lymphoma (DHL) is a rare and aggressive mature B-cell malignancy with concurrent MYC and BCL2 rearrangements. When DHL becomes relapsed or refractory, it becomes resistant to the majority of therapeutic approaches and has subpar clinical results. Therefore, innovative therapeutics for this particular patient population are urgently needed. METHODS: Orelabrutinib, a new oral BTK inhibitor, combined with the Bcl-2 inhibitor venetoclax, was used to confirm the antitumor effect of DHL. Cell counting kit-8 and Annexin V-FITC/PI assays were used to examine the interaction of this combined regimen on DHL cell lines and primary lymphoma cells. RNA sequencing, EdU incorporation assay, mitochondrial membrane potential assay, and western blotting were employed to explore the molecule mechanism for the cytotoxicity of orelabrutinib with or without venetoclax against DHL cell lines. RESULTS: In this study, orelabrutinib combined with venetoclax synergistically induced DHL cell death, as evidenced by the inhibition of cell proliferation, the induct of cell cycle arrest, and the promotion of cell apoptosis via the mitochondrial pathway. Orelabrutinib treatment alters genome-wide gene expression in DHL cells. The combined regimen decreases the expression of BTK and Mcl-1, potentially interfering with the activity and crosstalk of PI3K/AKT signaling and p38/MAPK signaling. In addition, the combination of orelabrutinib and venetoclax shows cytotoxic activity in primary B-lymphoma cells. CONCLUSION: In summary, these findings reveal a novel therapy targeting BCR signaling and the Bcl-2 family for DHL patients with a poor prognosis.

Therapeutic inhibition of PPARα-HIF1α-PGK1 signaling targets leukemia stem and progenitor cells in acute myeloid leukemia.

Treatment of acute myeloid leukemia (AML) with chemotherapeutic agents fails to eliminate leukemia stem cells (LSC)，and thus patients remain at high risk for relapse. Therefore, the identification of agents that target LSC is an important consideration for the development of new therapies. Enhanced glycolysis in LSC contributes to the aggressiveness of AML, which is difficult to be targeted. In this study, we showed that targeting peroxisome-proliferator-activated receptor α (PPARα), a ligand-activated transcription factor by chiglitazar provided a promising therapeutic approach. We first identified that chiglitazar reduced cell viability and proliferation of the leukemia stem-like cells population in AML. Treatment with chiglitazar blocked the ubiquitination of PPARα and increased its expression, resulting in the inhibition of glucose metabolism and apoptosis of AML cells. Consistent with its anti-leukemia stem-like cells activity in vitro, chiglitazar treatment in vivo resulted in the significant killing of leukemia stem-like cells as demonstrated in AML patient-derived xenograft (PDX) models. Mechanistically, PPARα overexpression inhibited the expression and promoter activity of PGK1 through blocking HIF1-α interaction on the PGK1 promoter. Thus, we concluded that targeting PPARα may serve as a novel approach for enhancing stem and progenitor cells elimination in AML.

Preclinical Evaluation of the Multiple Tyrosine Kinases Inhibitor Anlotinib in Leukemia Stem Cells.

Leukemia stem cells (LSCs) constitute the critical barrier to the cure of acute myeloid leukemia (AML) due to their chemoresistance and immune evasion property. Herein, the role of anlotinib, a multiple tyrosine kinase inhibitor, in killing LSCs and regulating chemoresistance and immune evasion was explored. Anlotinib treatment induced apoptosis of LSC-like cells as well as primary AML LSCs, while sparing the normal mononuclear cells in vitro. Moreover, anlotinib could impair the regeneration capacity of LSCs in the patient-derived leukemia xenograft mouse model. Mechanistically, anlotinib inhibited phosphorylation of c-kit, JAK2/STAT3, and STAT5, and downregulated STAT3 and STAT5 expression. In addition, anlotinib downregulated the anti-apoptotic proteins Bcl-2 and Bcl-xL, and upregulated Bax, thereby enhancing the sensitivity of LSCs to idarubicin in vitro. Intriguingly, anlotinib could also partially rescue the interferon-g production of T cells cocultured with LSCs by downregulating PD-L1 expression. In conclusion, anlotinib showed anti-LSC activity and the potential to enhance the sensitivity to idarubicin and inhibit the immunosuppressive feature of LSCs via JAK2/STAT signaling pathway downregulation in the preclinical study. Our results provided a rational basis for combinatory strategies involving anlotinib and chemotherapy or immunotherapy.

The electrochemiluminescence coreactant accelerator of metal-organic frameworks grafted with N-(aminobutyl)-N-(ethylisoluminol) for the ultrasensitive detection of chloramphenicol.

In this work, metal-organic frameworks (MOFs) are utilized as effective ECL coreactant accelerator to enhance the ECL responses of N-(aminobutyl)-N-(ethylisoluminol) (ABEI). Zn-based MOFs (MOF-Zn-1) were prepared by chelating Zn ions with melamine and thiophenedicarboxylic acid (TPDA), which observably accelerated the electrocatalytic oxidation of tripropylamine (TPA). Then, ABEI-MOF-Zn-1 as a high-performance ECL emitter was synthesized via an amide reaction between ABEI and mercaptopropionic acid (MPA) modified MOF-Zn-1. Strikingly, the ABEI-MOF-Zn-1 showed the 18-fold increase in the ECL signals relative to pure ABEI by using TPA as a coreactant. Moreover, ferrocene (Fc) as a quencher was first linked with capture DNA (cDNA), and then used to modify the ABEI-MOF-Zn-1, thereby constructing a label-free ECL biosensor. After the linkage between chloramphenicol (CAP) and aptamer DNA (aptDNA), the ECL response was definitely recovered by releasing L-DNA from double-stranded DNA (dsDNA, hybridization of aptDNA and L-DNA). The resultant sensor showed a wide linear range of 1.00 nM-0.10 mM (R2 = 0.99) and a low limit of detection (LOD) down to 0.11 nM for detecting CAP. This work developed a novel pattern to design an efficient ECL enhanced emitter, coupled by expanding its potential applications in clinical diagnosis.

Disulfiram/copper shows potent cytotoxic effects on myelodysplastic syndromes via inducing Bip-mediated apoptosis and suppressing autophagy.

Patients with myelodysplastic syndromes (MDS) who resist or fail to respond to hypomethylating agents (HMAs) show very poor outcomes and have no effective treatment strategies. Therefore, new therapeutic approaches are urgently needed for MDS patients harboring adverse prognostic factors. Repurposing disulfiram (DSF), an alcohol-abuse drug, with or without Copper (Cu) has attracted considerable attentions as a candidate anti-tumor therapy in diverse malignancies. However, the effect of DSF in the presence or absence of Cu on MDS has not been reported yet. In this study, we found that monotherapy with DSF showed mild cytotoxic effects on MDS preclinical models. However, the anti-tumor activity of DSF was significantly enhanced in the presence of Cu in MDS in vitro and in vivo with minimal safety profiles. DSF/Cu combination blocked MDS cell cycle progression at the G0/G1 phase, accompanied by reduction of the S phase. Accordingly, co-treatment with DSF and Cu downregulated the expression of Cyclin D1 and Cyclin A2, whereas this combination upregulated the level of P21 and P27. Mechanistically, the anti-MDS effectiveness of DSF/Cu was potentially associated with activation of the ER stress-related Bip pathway and inactivation of the Akt pathway. In addition, inhibition of autophagy process also contributed to the cytotoxicity of DSF/Cu in MDS cells. In conclusion, these findings provide impressive evidence that the DSF/Cu complex shows potent anti-tumor efficacies on MDS preclinical models, representing a potential alternative therapy for MDS patients and warranting further investigation in clinical contexts.

Low-Dose Triptolide Enhanced Activity of Idarubicin Against Acute Myeloid Leukemia Stem-like Cells Via Inhibiting DNA Damage Repair Response.

Leukemia stem cells (LSCs) are considered to be the root of relapse for acute myeloid leukemia (AML). Conventional chemotherapeutic drugs fail to eliminate LSCs. Therefore, new therapeutic strategies eliminating LSCs are urgently needed. Our results showed that low-dose Triptolide (TPL) enhanced the anti-AML activity of Idarubicin (IDA) in vitro against LSC-like cells (CD34 + CD38- KG1αand CD34 + CD38- kasumi-1 cells) and CD34+ primary AML cells, while sparing normal cells. Inspiringly, the combination treatment with low-dose TPL and IDA was also effective against CD34 + blasts from AML patients with FLT3-ITD mutation, which is an unfavorable risk factor for AML patients. Moreover, the combination of TPL and IDA induced a remarkable suppression of human leukemia growth in a xenograft mouse model. Mechanistically, the enhanced effect of low dose TPL on IDA against LSCs was attributed to inhibiting DNA damage repair response. Thus, our study may provide a theoretical basis to facilitate the development of a novel LSCs-targeting strategy for AML.Graphical abstract.

Quantitative determination of osteopontin in bovine, buffalo, yak, sheep and goat milk by Ultra-high performance liquid chromatography-tandem mass spectrometry and stable isotope dimethyl labeling.

Osteopontin (OPN) is a multifunctional protein present in different tissues, body fluids and milk. Different milk has different level of OPN content. To determine the amount of osteopontin in bovine, buffalo, yak, sheep and goat milk, we developed an ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method to detect an osteopontin signature peptide. The signature peptides selected by searching Uniprot database for trypsin digested osteopontin. The sample preparation procedure includes trypsin digestion, dimethyl labeling of tryptic peptides, purification and concentration of labeled tryptic peptide with solid phase extraction. The limit of detection and limit of quantification are 0.5 mg L-1 and 2.0 mg L-1, respectively. The method has satisfactory analytical performance with a linearity of R2 ≥ 0.998, recoveries of 103.7-111.0%, and precision of 1.8-6.2%. It is also validated and successfully applied to quantifying osteopontin content in bovine, buffalo, yak, sheep and goat milk.

Metabolic regulation of the bone marrow microenvironment in leukemia.

Most leukemia patients experience little benefit from immunotherapy, in part due to the immunosuppressive bone marrow microenvironment. Various metabolic mechanisms orchestrate the behaviors of immune cells and leukemia cells in the bone marrow microenvironment. Furthermore, leukemia cells regulate the bone marrow microenvironment through metabolism to generate an adequate supply of energy and to escape antitumor immune surveillance. Thus, the targeting of the interaction between leukemia cells and the bone marrow microenvironment provides a new therapeutic avenue. In this review, we describe the concept of the bone marrow microenvironment and several important metabolic processes of leukemia cells within the bone marrow microenvironment, including carbohydrate, lipid, and amino acid metabolism. In addition, we discuss how these metabolic pathways regulate antitumor immunity and reveal potential therapeutic targets.

T follicular helper cell-mediated IL-21 production suppresses FOXP3 expression of T follicular regulatory-like cells in diffuse large B cell lymphoma patients.

Based on CD25 expression, T follicular helper cells (Tfh) could be divided into T follicular regulatory (Tfr)-like subset (CD25+CD4+CXCR5+) and CD25- Tfh subset (CD25-CD4+CXCR5+). Patients with diffuse large B cell lymphoma (DLBCL) display high level of Tfr-like cells in blood and tumor. This Tfr-like subset could suppress CD8 T cell response while promote tumor cell proliferation. In this study, we investigated the transcription factors and regulatory elements associated with Tfr-like cells in DLBCL patients. Both circulating and tumor-infiltrating Tfr-like cells presented slightly higher Blimp-1 expression and significantly higher Foxp3 expression than the CD25- Tfh subset. As the IL-2 receptor, CD25 could be moderately upregulated in stimulated CD25- Tfh cells. However, stimulated CD25- Tfh cells could not upregulate Foxp3, indicating that the distinction between Foxp3-low CD25-CXCR5+CD4+ T cells and Foxp3-high CD25+CXCR5+CD4+ T cells was not due to differences in stimulation status. Regarding cytokine production, while both Tfr-like and CD25- Tfh cells upregulated IL-21 and IL-10 during stimulation, the CD25- Tfh cells presented significantly higher IL-21 and lower IL-10 expression than the Tfr-like cells, and the TGF-ß expression was only increased in Tfr-like cells. Interestingly, IL-21 secreted from CD25- Tfh cells negatively regulated the expression of Foxp3 and IL-10 of autologous Tfr-like cells. Together, these results demonstrated that the Tfr-like and CD25- Tfh subsets of circulating Tfh cells presented different functions and should be investigated separately.

A CpG Methylation Classifier to Predict Relapse in Adults with T-Cell Lymphoblastic Lymphoma.

PURPOSE: Adults with T-cell lymphoblastic lymphoma (T-LBL) generally benefit from treatment with acute lymphoblastic leukemia (ALL)-like regimens, but approximately 40% will relapse after such treatment. We evaluated the value of CpG methylation in predicting relapse for adults with T-LBL treated with ALL-like regimens. EXPERIMENTAL DESIGN: A total of 549 adults with T-LBL from 27 medical centers were included in the analysis. Using the Illumina Methylation 850K Beadchip, 44 relapse-related CpGs were identified from 49 T-LBL samples by two algorithms: least absolute shrinkage and selector operation (LASSO) and support vector machine-recursive feature elimination (SVM-RFE). We built a four-CpG classifier using LASSO Cox regression based on association between the methylation level of CpGs and relapse-free survival in the training cohort (n = 160). The four-CpG classifier was validated in the internal testing cohort (n = 68) and independent validation cohort (n = 321). RESULTS: The four-CpG-based classifier discriminated patients with T-LBL at high risk of relapse in the training cohort from those at low risk (P < 0.001). This classifier also showed good predictive value in the internal testing cohort (P < 0.001) and the independent validation cohort (P < 0.001). A nomogram incorporating five independent prognostic factors including the CpG-based classifier, lactate dehydrogenase levels, Eastern Cooperative Oncology Group performance status, central nervous system involvement, and NOTCH1/FBXW7 status showed a significantly higher predictive accuracy than each single variable. Stratification into different subgroups by the nomogram helped identify the subset of patients who most benefited from more intensive chemotherapy and/or sequential hematopoietic stem cell transplantation. CONCLUSIONS: Our four-CpG-based classifier could predict disease relapse in patients with T-LBL, and could be used to guide treatment decision.

Peptide Selection for Accurate Targeted Protein Quantification via a Dimethylation High-Resolution Mass Spectrum Strategy with a Peptide Release Kinetic Model.

A crucial step in accurate targeted protein quantification using targeted proteomics is to determine optimal proteotypic peptides representing targeted proteins. In this study, a workflow of peptide selection to determine proteotypic peptides using a dimethylation high-resolution mass spectrum strategy with a peptide release kinetic model was investigated and applied in peptide selection of bovine serum albumin. After specificity, digestibility, recovery, and stability evaluation of tryptic peptides in bovine serum albumin, the optimal proteotypic peptide was selected as LVNELTEFAK. The quantification method using LVNELTEFAK gave a linear range of 1-100 ppm with the coefficient greater than 0.9990, and the detection limit of bovine serum albumin in milk was 0.78 mg/kg. Compared with the proteotypic peptides selected by Skyline, the method showed a better performance in method validation. The workflow exhibited high comprehensiveness and efficiency in peptide selection, facilitating accurate targeted protein quantification in the food matrix, which lack protein standards.