Investigation of Age Gelation in UHT Milk

Abstract

Milk samples with twelve combinations of κ- and β-casein (CN) and β-lactoglobulin (β-Lg) variants were obtained to investigate the effect of protein variant on the mechanism/s of age gelation in ultra-high temperature (UHT) skim milk. Only milk groups with κ-CN/β-CN/β-Lg combinations AB/A1A2/AB and AB/A2A2/AB suffered from the expected age gelation over nine months storage, although this could not be attributed to the milk protein genetic variants. Top-down proteomics revealed three general trends across the twelve milk groups: (1) the abundance of intact native proteins decreases over storage time; (2) lactosylated proteoforms appear immediately post-UHT treatment; and (3) protein degradation products accumulate over storage time. Of the 151 identified degradation products, 106 (70.2%) arose from β-CN, 33 (21.9%) from αs1-CN, 4 (2.7%) from β-Lg, 4 (2.7%) from α-La, 3 (2%) from κ-CN and 1 (0.7%) from αs2-CN. There was a positive correlation between milk viscosity and 47 short peptides and four intact proteoforms, while 20 longer polypeptides and 21 intact proteoforms were negatively correlated. Age gelation was associated with specific patterns of proteolytic degradation and also with the absence of the families Bacillaceae, Aerococcaceae, Planococcaceae, Staphylococcaceae and Enterobacteriaceae, present in all the non-gelling milk groups pre-UHT.

Top-Down Proteomics of Medicinal Cannabis

“…In this study, we used a high number of microscans to perform spectral averaging in order to increase S/N, but the tradeoff was a longer duty cycle and acquisition time, which restricted throughput. In previous works, we employed a Q-ToF instrument that offered a spectral summation option and maximized S/N so much that very little background noise was left without overly prolonging the duty cycle [27][28][29]. Another advantage of the Q-ToF system is that it tolerated more complex samples without the need for SPE clean-up, which not only minimised the number of steps during sample preparation but also avoided protein loss.…”

“…In the first instance, a homemade database of 59 FASTA sequences [27][28][29] comprising horse myoglobin, all known allelic variants of bovine caseins, and the most abundant bovine whey proteins (α-lactalbumin, β-lactoglobulin, bovine serum albumin) was searched on our local Mascot server using a ±50 ppm fragment tolerance. The Mascot output is reported as a list of proteins and proteoforms in Supplementary Tables S2 and S3, respectively, as well as exemplified in Supplementary Figure S2A.…”

“…We previously published three studies on top-down proteomics of bovine milk samples using ultra-performance liquid chromatography (UPLC) online with a quadrupole time-of-flight (Q-ToF) mass spectrometer offering a 60,000 resolving power [27][28][29]. In these reports, major known milk proteins were targeted by first optimizing analytical methods on purchased standards and validating them on milk samples [28,29] and then applying the optimum method to a study aimed at exploring UHT milk (long life, ultra-heat treatment applied) properties during shelf-life storage [27]. Top-down sequencing information was obtained from CID and/or in-source CID experiments, which were the only fragmentation modes offered by this Q-ToF mass spectrometer.…”

“…Ultimately, we used the Genedata Expressionist software to develop workflows, allowing us to automate batch processing and annotation of both LC-MS and LC-MS/MS data [29]. This led to the discovery of extensive proteolysis of milk major proteins-in particular, beta-and alpha-S1-caseins-during the one-year storage of UHT milk [27]. Our experience on samples sourced from animals led us to tackling a top-down proteomics study on plant tissues.…”

Optimisation of Milk Protein Top-Down Sequencing Using In-Source Collision-Induced Dissociation in the Maxis Quadrupole Time-of-Flight Mass Spectrometer

“…In our laboratory, we host a maXis HD UHR-Q-ToF (60,000 resolution) with an ESI source on-line with a UHPLC 1290 Infinity Binary LC system (Agilent, Mulgrave, VIC, Australia), which we have used to develop a top-down method to analyse cow’s milk proteins [ 23 ]. This method was further implemented with CID MS/MS analysis and applied to the study of UHT milk shelf life [ 34 ]. The present study aimed at optimising MS/MS analysis by testing the effect of some of the parameters pertaining to IS-CID in combination or not with conventional CID.…”