Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This protocol provides step-by-step instructions for the development and application of quantitative assays using selected reaction monitoring (SRM) mass spectrometry (MS). First, likely quantotypic target peptides are identified based on numerous criteria. This includes identifying proteotypic peptides, avoiding sites of posttranslational modification, and analyzing the uniqueness of the target peptide to the target protein. Next, crude external peptide standards are synthesized and used to develop SRM assays, and the resulting assays are used to perform qualitative analyses of the biological samples. Finally, purified, quantified, heavy isotope labeled internal peptide standards are prepared and used to perform isotope dilution series SRM assays. Analysis of all of the resulting MS data is presented. This protocol was used to accurately assay the absolute abundance of proteins of the chemotaxis signaling pathway within RAW 264.7 cells (a mouse monocyte/macrophage cell line). The quantification of G_i2 (a heterotrimeric G-protein α-subunit) is described in detail.     

A Targeted Mass Spectrometry Strategy for Developing Proteomic Biomarkers: A Case Study of Epithelial Ovarian Cancer

1. Download : Download high-res image (120KB)
2. Download : Download full-size image

Highlights

• •Rigorous experimental design and data analysis for large-scale SRM studies.
• •Plasma-based biomarker signature combined with CA125 for ovarian cancer detection.
• •Broadly applicable strategy for the development of diagnostic biomarker assays.

     

Fatal primary diffuse large B‐cell lymphoma of the maxillary sinus initially treated as an infectious disease in an elderly patient: A clinicopathologic report

Objective

To report a case of primary diffuse large B‐cell lymphoma (DLBCL) of the maxillary sinus in an 82‐year‐old Caucasian woman.

Background

Diffuse large B‐cell lymphoma of the maxillary sinus has non‐specific signs and symptoms that may be confused with benign inflammatory conditions and upper respiratory infections.

Methods

An incisional biopsy was performed. CD20⁺/CD3^–/Ki‐67: 95% cells were observed.

Conclusion

A good medical history, clinical and imaging evaluations and immunohistochemical reactions are crucial to establish a correct and early diagnosis of DLBCL.     

inSPIRE: An Open-Source Tool for Increased Mass Spectrometry Identification Rates Using Prosit Spectral Prediction

Rescoring of mass spectrometry (MS) search results using spectral predictors can strongly increase peptide spectrum match (PSM) identification rates. This approach is particularly effective when aiming to search MS data against large databases, for example, when dealing with nonspecific cleavage in immunopeptidomics or inflation of the reference database for noncanonical peptide identification. Here, we present inSPIRE (in silico Spectral Predictor Informed REscoring), a flexible and performant open-source rescoring pipeline built on Prosit MS spectral prediction, which is compatible with common database search engines. inSPIRE allows large-scale rescoring with data from multiple MS search files, increases sensitivity to minor differences in amino acid residue position, and can be applied to various MS sample types, including tryptic proteome digestions and immunopeptidomes. inSPIRE boosts PSM identification rates in immunopeptidomics, leading to better performance than the original Prosit rescoring pipeline, as confirmed by benchmarking of inSPIRE performance on ground truth datasets. The integration of various features in the inSPIRE backbone further boosts the PSM identification in immunopeptidomics, with a potential benefit for the identification of noncanonical peptides.     

Decreased circulating miR-375: A potential biomarker for patients with non-small-cell lung cancer

MicroRNAs (miRNAs) are directly involved in cancer initiation, progression and metastasis. Alterations of miRNAs expression in cancer tissue may be reflected in circulation. We attempted to investigate the expression and clinical significance of plasma miR-20a, miR-31 and miR-375 in patients with non-small cell lung cancer (NSCLC). The plasma levels of miR-20a, miR-31 and miR-375 in 164 NSCLC patients and 164 healthy controls (discovery cohort) were evaluated and compared among various clinicopathological characteristics. The relationship between miRNA expression and clinical outcome of NSCLC patients was examined in an independent cohort (53 cases and 53 controls). The expression level of miR-375 in tissue was also examined. Plasma miR-375 levels in NSCLC patients were significantly decreased in both patient cohorts (P < 0.05). In addition, patients with metastatic NSCLC had lower plasma miR-375 expression than those with non-metastatic NSCLC (P < 0.05). Survival analysis showed that patients with low miR-375 expression had worse overall survival rates than those with high miR-375 expression (hazard ratios (HR) = 1.537 (1.046–2.258), P = 0.029). This association was independently validated in a separate cohort of 53 NSCLC patients (HR = 2.406, 95% CI 1.170–4.945, P = 0.017). The expression level of miR-375 was also found to be significantly down-regulated in NSCLC tissues compared with paracancerous tissues (P < 0.001). These findings indicate that miR-375 has an important role in NSCLC initiation and progression, and may be an independent poor prognostic factor in NSCLC patients.     

A laser ablation ICP‐MS based method for multiplexed immunoblot analysis: applications to manganese‐dependent protein dynamics of photosystem II in barley (Hordeum vulgare L.)

Manganese (Mn) constitutes an essential co‐factor in the oxygen‐evolving complex of photosystem II (PSII). Consequently, Mn deficiency reduces photosynthetic efficiency and leads to changes in PSII composition. In order to study these changes, multiplexed protein assays are advantageous. Here, we developed a multiplexed antibody‐based assay and analysed selected PSII subunits in barley (Hordeum vulgare L.). A selection of antibodies were labelled with specific lanthanides and immunoreacted with thylakoids exposed to Mn deficiency after western blotting. Subsequently, western blot membranes were analysed by laser ablation inductively coupled plasma mass spectrometry (LA‐ICP‐MS), which allowed selective and relative quantitative analysis via the different lanthanides. The method was evaluated against established liquid chromatography electrospray ionization tandem mass spectrometry (LC‐ESI‐MS/MS) methods, based on data‐dependent acquisition (DDA) and selected reaction monitoring (SRM). Manganese deficiency resulted in a general decrease in PSII protein abundances, an effect that was shown to be reversible upon Mn re‐supplementation. Specifically, the extrinsic proteins PsbP and PsbQ showed Mn‐dependent changes in abundances. Similar trends in the response to Mn deficiency at the protein level were observed when comparing DDA, SRM and LA‐ICP‐MS results. A biologically important exception to this trend was the loss of PsbO in the SRM analysis, which highlights the necessity of validating protein changes by more than one technique. The developed method enables a higher number of proteins to be multiplexed in comparison to existing immunoassays. Furthermore, multiplexed protein analysis by LA‐ICP‐MS provides an analytical platform with high throughput appropriate for screening large collections of plants.     

Urine as a source for clinical proteome analysis: From discovery to clinical application

The success of clinical proteome analysis should be assessed based on the clinical impact following implementation of findings. Although there have been several technological advancements in mass spectrometry in the last years, these have not resulted in similar advancements in clinical proteomics. In addition, application of proteomic biomarkers in clinical diagnostics and practical improvement in the disease management is extremely rare. In this review, we discuss the relevant issues associated with identification of robust biomarkers of clinical value. Urine appears to be an ideal source of biomarkers, for theoretical, methodological, and practical reasons. Therefore, this review is focused on the search for biomarkers in urine within the last decade. Urine can be used for non-invasive assessment of a variety of diseases including those affecting the urogenital tract and also other pathologies such as cardiovascular disease or appendicitis. We also discuss the importance of data validation, an essential step in translating biomarkers into the clinical practice. Furthermore, we examine several examples of apparently successful proteomic biomarker discovery studies and their implications for disease diagnosis, prognosis, and therapy evaluation. We also discuss some current challenges in this field and reflect on future research prospects. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.     

The cancer cell secretome: A good source for discovering biomarkers?

Cancer is a leading cause of death. Early detection is usually associated with better clinical outcomes. Recent advances in genomics and proteomics raised hopes that new biomarkers for diagnosis, prognosis or monitoring therapeutic response will soon be discovered. Proteins secreted by cancer cells, referred also as “the cancer cell secretome”, is a promising source for biomarker discovery. In this review we will summarize recent advances in cancer cell secretome analysis, focusing on the five most fatal cancers (lung, breast, prostate, colorectal, and pancreatic). For each cancer type we will describe the proteomic approaches utilized for the identification of novel biomarkers. Despite progress, identification of markers that are superior to those currently used has proven to be a difficult task and very few, if any, newly discovered biomarker has entered the clinic the last 10 years.     

Cell polarization: From epithelial cells to odontoblasts

Cell polarity identifies the asymmetry of a cell. Various types of cells, including odontoblasts and epithelial cells, polarize to fulfil their destined functions. Odontoblast polarization is a prerequisite and fundamental step for tooth development and tubular dentin formation. Current knowledge of odontoblast polarization, however, is very limited, which greatly impedes the development of novel approaches for regenerative endodontics. Compared to odontoblasts, epithelial cell polarization has been extensively studied over the last several decades. The knowledge obtained from epithelia polarization has been found applicable to other cell types, which is particularly useful considering the remarkable similarities of the morphological and compositional features between polarized odontoblasts and epithelia. In this review, we first discuss the characteristics, the key regulatory factors, and the process of epithelial polarity. Next, we compare the known facts of odontoblast polarization with epithelial cells. Lastly, we clarify knowledge gaps in odontoblast polarization and propose the directions for future research to fill the gaps, leading to the advancement of regenerative endodontics.     

Proteomic analysis of B-cell malignancies

The identification of proteins aberrantly expressed in malignant B-cells can potentially be used to develop new diagnostic, prognostic or therapeutic targets. Proteomic studies of B-cell malignancies have made significant progress, but further studies are needed to increase our coverage of the B-cell malignant proteome. To achieve this goal we stress the advantages of using sub-cellular fractionation, protein separation, quantitation and affinity purification techniques to identify hitherto unidentified signalling and regulatory proteins. For example, proteomic analysis of B-cell plasma membranes isolated from patients with mantle cell lymphoma (MCL) identified the voltage-gated proton channel (HVCN1,[1]). This protein has now been characterised as a key modulator of B-cell receptor (BCR) signalling and abrogation of HVCN1 function could have a role in the treatment of B-cell malignancies dependent on maintained BCR signalling [2]. Similarly, proteomic studies on cell lysates from prognostic subtypes of CLL, distinguished by the absence (UM-CLL) or presence (M-CLL) of somatic hypermutation of the immunoglobulin heavy chain locus identified nucleophosmin 1 (NMP1) as a potential prognostic marker [3,4]. Thus, targeted proteomic analysis on selected organelles or sub-cellular compartments can identify novel proteins with unexpected localisation or function in malignant B-cells that could be developed for clinical purposes.