Journal of Mass Spectrometry and Advances in the Clinical Lab最新文献

Introduction

Human saliva contains a wealth of proteins that can be monitored for disease diagnosis and progression. Saliva, which is easy to collect, has been extensively studied for the diagnosis of numerous systemic and infectious diseases. However, the presence of amylase, the most abundant protein in saliva, can obscure the detection of low-abundance proteins by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-ToF MS), thus reducing its diagnostic utility.

Objectives

In this study, we used a device to deplete salivary amylase from water-gargle samples by affinity adsorption. Following depletion, saliva proteome profiling was performed using MALDI-ToF MS on gargle samples from individuals confirmed to have COVID-19 based on nasopharyngeal (NP) swab reverse transcription quantitative polymerase chain reaction (RT-qPCR).

Results

The depletion of amylase led to increased signal intensities of various peaks and the detection of previously unobserved peaks in the MALDI-ToF MS spectra. The overall specificity and sensitivity after amylase depletion were 100% and 85.17%, respectively, for detecting COVID-19.

Conclusion

This simple, rapid, and inexpensive technique for depleting salivary amylase can reveal spectral diversity in saliva using MALDI-ToF MS, expose low-abundance proteins, and assist in establishing novel biomarkers for diseases.

Objective

To develop and validate an assay for the analysis of bedaquiline and its M2 metabolite in human breast milk.

Methods

The analytes were extracted using solid phase extraction following protein precipitation. Quantification was performed with liquid chromatography coupled with tandem mass spectrometry. Chromatographic separation was achieved using gradient chromatography on a Poroshell 120 SB-C18 analytical column at 40 °C, with a flow rate of 350 µL/minute and a total run time of eight minutes. An AB Sciex 3000 mass spectrometer with electrospray ionization in the positive mode was used for detection, employing multiple reaction monitoring scan mode. Bedaquiline-d6 and M2-d3-¹³C were used as internal standards.

Results

Calibrations curves for bedaquiline and M2 exhibited quadratic (weighted 1/x concentration) regressions over the respective concentration ranges of 0.0780 to 5.00 µg/mL and 0.0312 to 2.00 µg/mL. Inter- and intra-day validation accuracies ranged between 96.7 % and 103.5 % for bedaquiline, and 104.2 % to 106.5 % for M2, with a coefficient of variation below 9.2 % for both compounds.

Conclusion

The developed assay demonstrated selectivity and robustness, enabling differentiation between bedaquiline and M2 within the context of endogenous compounds from six separate lots of breast milk samples. Successful application was observed in the analysis of breast milk samples sourced from patients treated for multidrug-resistant tuberculosis within a clinical study setting.

Background

Measurement of trough levels for calcineurin inhibitors by venipuncture sampling is a mainstay of patient management in solid organ transplant recipients but challenging in pediatric patients. Volumetric Absorptive Microsampling (VAMS) is a patient-friendly, minimally invasive sampling technique to accurately collect blood. An assay for measurement of tacrolimus in blood using VAMS, coupled with parallel reaction monitoring (PRM) mass spectrometry, was validated in pediatric heart transplant patients.

Methods

Tacrolimus was measured by a newly developed high-resolution PRM assay and compared with low-resolution tandem mass spectrometry (MRM). Dried blood samples were collected from pediatric heart transplant patients (n = 35) using VAMS devices and a satisfaction survey was completed by patients/guardians. Tacrolimus concentrations were compared across whole liquid blood, dried blood spots, and capillary blood, and shipping stability determined.

Results

The PRM assay was linear over a range 1–50 ng/mL, similar to MRM but had greater specificity due to reduced background noise. No significant differences in tacrolimus concentrations were observed between VAMS and venous blood. Tacrolimus dried on VAM tips was stable for 14 days and concentrations were unaffected by postal shipping. The variability in two simultaneously collected at-home patient samples was minimal – average concentration difference was 0.12 ± 0.94 ng/mL (p = 0.6) between paired samples.

Conclusion

A high resolution PRM mass spectrometry assay was developed for home-based dried blood collections for therapeutic monitoring of tacrolimus. The advantage of PRM was enhanced specificity and the VAMS devices provided a simple and convenient approach to blood sampling at home in pediatric heart transplant patients.

Introduction

LC-MS-based methods for protein quantification have a stigma of being relatively expensive and low-throughput. This is partly due to the cost and speed of trypsin digestion, which has primarily focused on advancements in research-based biomarker discovery applications that rely on protein/peptide identifications rather than clinical biomarker quantification. However, there is a need for simple, fast, and reproducibly efficient surrogate peptide recovery in clinical biomarker quantification.

Methods

Multiple methodologies were evaluated to enhance tryptic digestion for the analysis of thyroglobulin, a prototypical serum protein biomarker. The main criteria for assessment were the yield and speed of formation of surrogate peptides. Various factors such as different additives, types of trypsin, microwave- and pressure-assisted systems, and enzyme concentration were considered as key variables, in addition to digestion time.

Results

It was observed that digestion additives/denaturants had a significant impact on the speed and yield of digestion for each surrogate peptide. Increasing the concentration of trypsin alone was found to accelerate digestions appreciably for most surrogate peptides, without affecting the yield. However, the use of sequencing-grade trypsins and microwave/pressure-assisted systems did not offer significant advantages over the use of 'standard-grade' TPCK-treated trypsin in combination with a conventional incubator, once digestion time and additive had been optimized.

Conclusion

We have dispelled the notion that trypsin digestion is inherently slow and expensive for targeted quantification of serum proteins. Additionally, we have established a groundwork for experimentation that can pave the way for the creation of efficient trypsin digestion protocols, aiming to optimize yield, speed, and cost. It is our hope that these advancements will promote the wider incorporation of such assays in clinical laboratories.

Introduction

The VeriStrat® test (VS) is a blood-based assay that predicts a patient's response to therapy by analyzing eight features in a spectrum obtained from matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) analysis of human serum and plasma. In a recent analysis of the INSIGHT clinical trial (NCT03289780), it was found that the VS labels, VS Good and VS Poor, can effectively predict the responsiveness of non-small cell lung cancer (NSCLC) patients to immune checkpoint inhibitor (ICI) therapy. However, while VS measures the intensities of spectral features using MALDI-TOF analysis, the specific proteoforms underlying these features have not been comprehensively identified.

Objectives

The objective of this study was to identify the proteoforms that are measured by VS.

Methods

To resolve the features obtained from the low-resolution MALDI-TOF procedure used to acquire mass spectra for VS DeepMALDI® analysis of serum was employed. This technique allowed for the identification of finer peaks within these features. Additionally, a combination of reversed-phase fractionation and liquid chromatography-tandem mass spectrometry (LC-MS/MS) was then used to identify the proteoforms associated with these peaks.

Results

The analysis revealed that the primary constituents of the spectrum measured by VS are serum amyloid A1, serum amyloid A2, serum amyloid A4, C-reactive protein, and beta-2 microglobulin.

Conclusion

Proteoforms involved in host immunity were identified as significant components of these features. This newly acquired information improves our understanding of how VS can accurately predict patient response to therapy. It opens up additional studies that can expand our understanding even further.

Background and aims

Reliable lead screening methods are necessary to support early identification of lead exposure in children. Sample collection using dried blood spots (DBS) offers advantages compared to traditional venipuncture and capillary collection. Here, we describe and compare three lead DBS inductively coupled plasma-mass spectrometry (ICP-MS) methods for lead screening.

Materials and methods

Lead was extracted from Whatman 903 protein saver cards punches and analyzed by ICP-MS across three independent clinical laboratories. Each laboratory evaluated the performance of aqueous and matrix-matched DBS calibrators using external quality control samples (WI State of Laboratory of Hygiene Program). Leftover patient samples (n = 39) were used for an interlaboratory comparison of lead DBS. Lead DBS results were compared to whole blood methods.

Results

The DBS ICP-MS methods using matrix-matched DBS calibrators had superior performance to the aqueous calibrations. There was a strong correlation between lead measured in DBS (matrix-matched) and whole blood for the three methods evaluated.

Conclusion

Lead can be measured accurately by ICP-MS in DBS samples when matrix-matched calibrators are used. External quality control programs are valuable to assess the performance of DBS methods. DBS lead ICP-MS methods are a robust analytical option for lead screening even though the limitations of DBS are well recognized.

Introduction

Steroid measurements are important for diagnosis and monitoring of many conditions and treatment regiments; however, due to structural and chemical similarities amongst steroids, these analyses are challenging, even for highly specific techniques such as liquid chromatography-tandem mass spectrometry (LC-MS/MS). Differential mobility spectrometry (DMS) has the potential to improve these analyses by providing an orthogonal and complementary separation technique.

Methods

Initially, the potential for DMS to improve signal-to-noise ratio (S/N) and reduce interference was tested by comparing chromatograms acquired with and without DMS when performing measurements of six different steroids. Subsequently, a full clinical validation of cortisol and cortisone in urine was performed with the LC-DMS-MS/MS method.

Results and Discussion

DMS significantly reduced interferences observed in the chromatograms and boosted S/N by between 1.6 and 13.8 times. Additionally, DMS improved the agreement between quantifier/qualifier fragment ion results for cortisol and cortisone as indicated by the increase in R² from approximately 0.81 to 0.98. All validation studies met acceptance criteria and we observed exceptional analytical performance in terms of precision, with % CVs less than 8%.

Conclusions

DMS improved the specificity of the steroid measurements by reducing interferences and improving S/N. The validation studies prove that these benefits did not come at the expense of other aspects of analytical performance. This study indicates that DMS has the potential to benefit not just clinical measurements of challenging analytes, but many clinical LC-MS/MS analyses.

Pipettes are essential tools for biomedical and analytical laboratories, analogous to workstations for computer scientists. Variation in pipetting is a known unknown, as it is generally accepted that variations exist, but thus far, there have been limited studies on the extent of these variations in practice. In this mini-review, we highlight how manual pipetting is a key technique in the laboratory, and, although simple, inaccuracy and imprecision exist. If variations are not adequately addressed, errors can be compounded and consequently compromise data quality. Determination of the accuracy and precision of manual pipetting is straightforward, and here we review two common approaches that use gravimetry and spectrophotometry as readouts. We also provide detailed protocols for determination of accuracy and precision using manual single and multi-channel pipettes. These simple-to-use methods can be used by any laboratory for competency training and regular checks. Having a common protocol for evaluation of variation will also enable cross-laboratory comparison and potentially facilitate establishment of a reference value of acceptable ranges for operator error. Such a value could be of relevance to the scientific community for benchmarking and assuring good laboratory practice.