Cow's milk is an important source of proteins in human nutrition. On average, cow's milk contains 3.5% protein. The most abundant proteins in bovine milk are caseins and some of the whey proteins, ...namely beta-lactoglobulin, alpha-lactalbumin, and serum albumin. A number of allelic variants and post-translationally modified forms of these proteins have been identified. Their occurrence varies with breed, individuality, stage of lactation, and health and nutritional status of the animal. It is therefore essential to have reliable methods of detection and quantitation of these proteins. Traditionally, major milk proteins are quantified using liquid chromatography (LC) and ultra violet detection method. However, as these protein variants co-elute to some degree, another dimension of separation is beneficial to accurately measure their amounts. Mass spectrometry (MS) offers such a tool. In this study, we tested several RP-HPLC and MS parameters to optimise the analysis of intact bovine proteins from milk. From our tests, we developed an optimum method that includes a 20-28-40% phase B gradient with 0.02% TFA in both mobile phases, at 0.2 mL/min flow rate, using 75°C for the C8 column temperature, scanning every 3 sec over a 600-3000 m/z window. The optimisations were performed using external standards commercially purchased for which ionisation efficiency, linearity of calibration, LOD, LOQ, sensitivity, selectivity, precision, reproducibility, and mass accuracy were demonstrated. From the MS analysis, we can use extracted ion chromatograms (EICs) of specific ion series of known proteins and integrate peaks at defined retention time (RT) window for quantitation purposes. This optimum quantitative method was successfully applied to two bulk milk samples from different breeds, Holstein-Friesian and Jersey, to assess differences in protein variant levels.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Epithelial ovarian cancer is one of the most fatal gynecological malignancies in adult women. As studies on protein N‐glycosylation have extensively reported aberrant patterns in the ovarian cancer ...tumor microenvironment, obtaining spatial information will uncover tumor‐specific N‐glycan alterations in ovarian cancer development and progression. matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is employed to investigate N‐glycan distribution on formalin‐fixed paraffin‐embedded ovarian cancer tissue sections from early‐ and late‐stage patients. Tumor‐specific N‐glycans are identified and structurally characterized by porous graphitized carbon‐liquid chromatography‐electrospray ionization‐tandem mass spectrometry (PGC‐LC‐ESI‐MS/MS), and then assigned to high‐resolution images obtained from MALDI‐MSI. Spatial distribution of 14 N‐glycans is obtained by MALDI‐MSI and 42 N‐glycans (including structural and compositional isomers) identified and structurally characterized by LC‐MS. The spatial distribution of oligomannose, complex neutral, bisecting, and sialylated N‐glycan families are localized to the tumor regions of late‐stage ovarian cancer patients relative to early‐stage patients. Potential N‐glycan diagnostic markers that emerge include the oligomannose structure, (Hex)6 + (Man)3(GlcNAc)2, and the complex neutral structure, (Hex)2 (HexNAc)2 (Deoxyhexose)1 + (Man)3(GlcNAc)2. The distribution of these markers is evaluated using a tissue microarray of early‐ and late‐stage patients.
Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS), in combination with Biotyper software, is a rapid, high-throughput, and accurate method for the ...identification of microbes. Microbial outbreaks in a brewery present a major risk for companies as it can lead to cost-intensive recalls and damage to the brand reputation. MALDI-TOF MS has been implemented into a brewery setting for quality control practices and the identification of beer spoilage microorganisms. However, the applicability of this approach is hindered by compatibility issues associated with mixed cultures, requiring the use of time-consuming selective cultivation techniques prior to identification. We propose a novel, low-cost approach based on the combination of inertial microfluidics and secondary flows in a spiral microchannel for high-throughput and efficient separation of yeasts (
Saccharomyces pastorianus
and
Saccharomyces cerevisiae
) from beer spoilage microorganisms (
Lactobacillus brevis
and
Pediococcus damnosus
). Flow rates were optimised using
S. pastorianus
and
L. brevis
, leading to separation of more than 90% of the
L. brevis
cells from yeast. The microorganisms were then identified to the species level using the MALDI-TOF MS platform using standard sample preparation protocols. This study shows the high-throughput and rapid separation of spoilage microorganisms (0.3-3 μm) from background yeast (5 μm) from beer, subsequent identification using MALDI Biotyper, and the potential applicability of the approach for biological control in the brewing industry.
Microfluidics and MALDI-TOF MS is a rapid, high-throughput, and accurate method for the identification of beer spoilage bacteria.
Recent developments in spatial proteomics have paved the way for retrospective in situ mass spectrometry (MS) analyses of formalin-fixed paraffin-embedded clinical tissue samples. This type of ...analysis is commonly referred to as matrix-assisted laser desorption/ionization (MALDI) imaging. Recently, formalin-fixed paraffin-embedded MALDI imaging analyses were augmented to allow in situ analyses of tissue-specific
N
-glycosylation profiles. In the present study, we outline an improved automated sample preparation method for
N
-glycan MALDI imaging, which uses in situ PNGase F-mediated release and measurement of
N
-linked glycans from sections of formalin-fixed murine kidney. The sum of the presented data indicated that
N
-glycans can be cleaved from proteins within formalin-fixed tissue and characterized using three strategies: (i) extraction and composition analysis through on-target MALDI MS and liquid chromatography coupled to electrospray ionization ion trap MS; (ii) MALDI profiling, where
N
-glycans are released and measured from large droplet arrays in situ; and (iii) MALDI imaging, which maps the tissue specificity of
N
-glycans at a higher resolution. Thus, we present a complete, straightforward method that combines MALDI imaging and characterization of tissue-specific
N
-glycans and complements existing strategies.
Graphical Abstract
MALDI imaging MS of N-linked glycans released from formalin-fixed paraffin-embedded murine kidney sections. Ion intensity maps for (Hex)
2
(HexNAc)
3
(Deoxyhexose)
3
+(Man)
3
(GlcNAc)
2
(
m/z
2304.932, red), (Hex)
6
+(Man)
3
(GlcNAc)
2
(
m/z
1905.742, green) and (Hex)
2
(HexNAc)
2
+(Man)
3
(GlcNAc)
2
(
m/z
1663.756, blue)
Leukocyte homing driven by the chemokine CCL21 is pivotal for adaptive immunity because it controls dendritic cell (DC) and T cell migration through CCR7. ACKR4 scavenges CCL21 and has been shown to ...play an essential role in DC trafficking at the steady state and during immune responses to tumors and cutaneous inflammation. However, the mechanism by which ACKR4 regulates peripheral DC migration is unknown, and the extent to which it regulates CCL21 in steady-state skin and lymph nodes (LNs) is contested. Specifically, our previous findings that CCL21 levels are increased in LNs of ACKR4-deficient mice I. Comerford et al.,
116, 4130-4140 (2010) were refuted M. H. Ulvmar et al.,
15, 623-630 (2014), and no differences in CCL21 levels in steady-state skin of ACKR4-deficient mice were reported despite compromised CCR7-dependent DC egress in these animals S. A. Bryce et al.,
196, 3341-3353 (2016). Here, we resolve these issues and reveal that two forms of CCL21, full-length immobilized and cleaved soluble CCL21, exist in steady-state barrier tissues, and both are regulated by ACKR4. Without ACKR4, extracellular CCL21 gradients in barrier sites are saturated and nonfunctional, DCs cannot home directly to lymphatic vessels, and excess soluble CCL21 from peripheral tissues pollutes downstream LNs. The results identify the mechanism by which ACKR4 controls DC migration in barrier tissues and reveal a complex mode of CCL21 regulation in vivo, which enhances understanding of functional chemokine gradient formation.
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•LC-MS is an analytical technique well suited to measuring proteins in complex samples.•There is limited application of LC-MS to measuring cattle proteins for quantifying ...phenotypes.•Accurately measuring β-casein proteoforms in cows’ milk remains elusive.
Modern mass spectrometers can accurately measure thousands of compounds in complex mixtures over a given liquid chromatograph method, depending on desired outcome and method duration. This stream of analytical chemistry has wide ranging application across food, pharma, environmental, forensics, clinical and research. With consistent pressure on both the ruminant production and product industries to face new and substantial challenges, liquid chromatography-mass spectrometry (LC-MS) is an ideal tool to identify, detect and quantify markers of breeding, production and adaption to support both research and industry to overcome these challenges. Herein, we provide a description of the theoretical basis and framework for LC-MS as a rapidly developing technique and highlight its application in measuring cattle and cattle product traits through protein quantitation with specific focus on beta-casein proteoforms.
Matrix assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) can determine the spatial distribution of analytes such as protein distributions in a tissue section according to ...their mass-to-charge ratio. Here, we explored the clinical potential of machine learning (ML) applied to MALDI MSI data for cancer diagnostic classification using tissue microarrays (TMAs) on 302 colorectal (CRC) and 257 endometrial cancer (EC)) patients. ML based on deep neural networks discriminated colorectal tumour from normal tissue with an overall accuracy of 98% in balanced cross-validation (98.2% sensitivity and 98.6% specificity). Moreover, our machine learning approach predicted the presence of lymph node metastasis (LNM) for primary tumours of EC with an accuracy of 80% (90% sensitivity and 69% specificity). Our results demonstrate the capability of MALDI MSI for complementing classic histopathological examination for cancer diagnostic applications.
The strength of MALDI-MSI is to analyze and visualize spatial intensities of molecular features from an intact tissue. The distribution of the intensities can then be visualized within a single ...tissue section or compared in between sections, acquired consecutively. This method can be reliably used to reveal physiological structures and has the potential to identify molecular details, which correlate with biological outcomes. MALDI-MSI implementation in clinical laboratories requires the ability to ensure method quality and validation to meet diagnostic expectations. To be able to get consistent qualitative and quantitative results, standardized sample preparation and data acquisition are of highest priority. We have previously shown that the deposition of internal standards onto the tissue section during sample preparation can be used to improve the mass accuracy of monitored m/z features across the sample. Here, we present the use of external and internal controls for the quality check of sample preparation and data acquisition, which is particularly relevant when either many spectra are acquired during a single MALDI-MSI experiment or data from independent experiments are processed together. To monitor detector performance and sample preparation, we use egg white as an external control for peptide and N-glycan MALDI-MSI throughout the experiment. We have also identified endogenous peptides from cytoskeletal proteins, which can be reliably monitored in gynecological tissue samples. Lastly, we summarize our standard quality control workflow designed to produce reliable and comparable MALDI-MSI data from single sections and tissue microarrays (TMAs).
N
-Glycan alterations contribute to the pathophysiology and progression of various diseases. However, the involvement of
N
-glycans in knee osteoarthritis (KOA) progression at the tissue level, ...especially within articular cartilage, is still poorly understood. Thus, the aim of this study was to spatially map and identify KOA-specific
N
-glycans from formalin-fixed paraffin-embedded (FFPE) osteochondral tissue of the tibial plateau relative to cadaveric control (CTL) tissues. Human FFPE osteochondral tissues from end-stage KOA patients (
n
=3) and CTL individuals (
n
=3), aged >55 years old, were analyzed by matrix‐assisted laser desorption/ionization mass spectrometry imaging (MALDI‐MSI) and liquid chromatography–tandem mass spectrometry (LC-MS/MS). Overall, it was revealed that 22
N
-glycans were found in the cartilage region of KOA and CTL tissue. Of those, 15
N
-glycans were more prominent in KOA cartilage than CTL cartilage. We then compared sub-regions of KOA and CTL tissues based on the Osteoarthritis Research Society International (OARSI) histopathological grade (1 to 6), where 1 is an intact cartilage surface and 6 is cartilage surface deformation. Interestingly, three specific complex-type
N
-glycans, (Hex)
4
(HexNAc)
3
, (Hex)
4
(HexNAc)
4
, and (Hex)
5
(HexNAc)
4
, were found to be localized to the superficial fibrillated zone of degraded cartilage (KOA OARSI 2.5-4), compared to adjacent cartilage with less degradation (KOA OARSI 1-2) or relatively healthy cartilage (CTL OARSI 1-2). Our results demonstrate that
N
-glycans specific to degraded cartilage in KOA patients have been identified at the tissue level for the first time. The presence of these
N
-glycans could further be evaluated as potential diagnostic and prognostic biomarkers.
Osteoarthritis (OA) is the most common degenerative joint disease, predicted to increase in incidence year by year due to an ageing population. Due to the biological complexity of the disease, OA ...remains highly heterogeneous. Although much work has been undertaken in the past few years, underlying molecular mechanisms leading to joint tissue structural deterioration are not fully understood, with only few validated markers for disease diagnosis and progression being available. Discovery and quantitation of various OA-specific biomarkers is still largely focused on the bodily fluids which does not appear to be reliable and sensitive enough. However, with the advancement of spatial proteomic techniques, several novel peptides and proteins, as well as N-glycans, can be identified and localised in a reliable and sensitive manner. To summarise the important findings from OA biomarker studies, papers published between 2000 and 2020 were searched via Google Scholar and PubMed. Medical subject heading (MeSH) terms 'osteoarthritis', 'biomarker', 'synovial fluid', 'serum', 'urine', 'matrix-assisted laser desorption/ionisation', 'mass spectrometry imaging', 'proteomic', 'glycomic', 'cartilage', 'synovium' AND 'subchondral bone' were selectively used. The literature search was restricted to full-text original research articles and written only in English. Two main areas were reviewed for OA biomarker studies: (1) an overview of disease-specific markers detected from different types of OA bio-samples, and (2) an up-to-date summary of the tissue-specific OA studies that have utilised matrix-assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI). Overall, these OA biomarkers could provide clinicians with information for better the diagnosis, and prognosis of individual patients, and ultimately help facilitate the development of disease-modifying treatments.
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