Karlie A. Neilson

Less label, more free: Approaches in label-free quantitative mass spectrometry

In this review we examine techniques, software, and statistical analyses used in label‐free quantitative proteomics studies for area under the curve and spectral counting approaches. Recent advances in the field are discussed in an order that reflects a logical workflow design. Examples of studies that follow this design are presented to highlight the requirement for statistical assessment and further experiments to validate results from label‐free quantitation. Limitations of label‐free approaches are considered, label‐free approaches are compared with labelling techniques, and forward‐looking applications for label‐free quantitative data are presented. We conclude that label‐free quantitative proteomics is a reliable, versatile, and cost‐effective alternative to labelled quantitation.

Proteomic analysis of temperature stress in plants

In this review we examine current approaches used for proteomic analysis of temperature stress in plants. Rapid advances in this field in recent years are discussed, including metabolic, chemical and isotopic labeling, and label‐free quantitative techniques. These are compared and contrasted with well‐established methods such as 2‐DE approaches. Examples of applications of various methods are presented, and technical difficulties and limitations of each are also considered. Results of previous studies are examined in detail, and commonly occurring temperature stress response proteins are collated. We conclude that technical advances, and improvements in genome sequence availability, will have an ever increasing impact on our understanding of molecular mechanisms of stress response in plants.

Quantitative proteomic analysis of cold-responsive proteins in rice

Rice is susceptible to cold stress and with a future of climatic instability we will be unable to produce enough rice to satisfy increasing demand. A thorough understanding of the molecular responses to thermal stress is imperative for engineering cultivars, which have greater resistance to low temperature stress. In this study we investigated the proteomic response of rice seedlings to 48, 72 and 96 h of cold stress at 12–14°C. The use of both label‐free and iTRAQ approaches in the analysis of global protein expression enabled us to assess the complementarity of the two techniques for use in plant proteomics. The approaches yielded a similar biological response to cold stress despite a disparity in proteins identified. The label‐free approach identified 236 cold‐responsive proteins compared to 85 in iTRAQ results, with only 24 proteins in common. Functional analysis revealed differential expression of proteins involved in transport, photosynthesis, generation of precursor metabolites and energy; and, more specifically, histones and vitamin B biosynthetic proteins were observed to be affected by cold stress.

Thermal Stability of Thaumatin-Like Protein, Chitinase, and Invertase Isolated from Sauvignon blanc and Semillon Juice and Their Role in Haze Formation in Wine

A thermal unfolding study of thaumatin-like protein, chitinase, and invertase isolated from Vitis vinifera Sauvignon blanc and Semillon juice was undertaken. Differential scanning calorimetry demonstrated that chitinase was a major player in heat-induced haze in unfined wines as it had a low melt temperature, and aggregation was observed. The kinetics of chitinase F1 (Sauvignon blanc) unfolding was studied using circular dichroism spectrometry. Chitinase unfolding conforms to Arrhenius behavior having an activation energy of 320 kJ/mol. This enabled a predictive model for protein stability to be generated, predicting a half-life of 9 years at 15 degrees C, 4.7 days at 30 degrees C, and 17 min at 45 degrees C. Circular dichroism studies indicate that chitinase unfolding follows three steps: an initial irreversible step from the native to an unfolded conformation, a reversible step between a collapsed and an unfolded non-native conformation, followed by irreversible aggregation associated with visible haze formation.

Roles of grape thaumatin-like protein and chitinase in white wine haze formation

Grape chitinase was found to be the primary cause of heat-induced haze formation in white wines. Chitinase was the dominant protein in a haze induced by treating Sauvignon blanc wine at 30 °C for 22 h. In artificial wines and real wines, chitinase concentration was directly correlated to the turbidity of heat-induced haze formation (50 °C for 3 h). Sulfate was confirmed to have a role in haze formation, likely by converting soluble aggregates into larger visible haze particles. Thaumatin-like protein was detected in the insoluble fraction by SDS-PAGE analysis but had no measurable impact on turbidity. Differential scanning calorimetry demonstrated that the complex mixture of molecules in wine plays a role in thermal instability of wine proteins and contributes additional complexity to the wine haze phenomenon.

Two-step purification of pathogenesis-related proteins from grape juice and crystallization of thaumatin-like proteins

Grape thaumatin-like (TL) proteins and chitinases play roles in plant-pathogen interactions and can cause protein haze in white wine unless removed prior to bottling. A two-step method is described that highly purified hundreds of milligrams of TL proteins and chitinases from two juices by strong cation exchange (SCX) and hydrophobic interaction chromatography (HIC). The method was fast and separated isoforms of TL proteins and chitinases from within the same juice, in most cases to >97% purity. The isolated proteins were identified by peptide nanoLC-MS/MS and crystallized using a high-throughput screening method. Crystals from three protein fractions produced high-resolution X-ray crystallography data.

Label-Free Quantitative Shotgun Proteomics Using Normalized Spectral Abundance Factors

In this chapter we describe the workflow used in our laboratory for label-free quantitative shotgun proteomics based on spectral counting. The main tools used are a series of R modules known collectively as the Scrappy program. We describe how to go from peptide to spectrum matching in a shotgun proteomics experiment using the XTandem algorithm, to simultaneous quantification of up to thousands of proteins, using normalized spectral abundance factors. The outputs of the software are described in detail, with illustrative examples provided for some of the graphical images generated. While it is not strictly within the scope of this chapter, some consideration is given to how best to extract meaningful biological information from quantitative shotgun proteomics data outputs.

The influence of signals from chilled roots on the proteome of shoot tissues in rice seedlings

Low root temperature causes a decrease in water uptake, which leads to mineral and nutrient deficiencies with potentially decreased root and shoot growth. Differential temperature effects in plants have been studied extensively, however, the effect of root chilling on the global protein expression in shoots has not been explored. In this study, we imposed chilling temperatures on roots of rice plants while maintaining shoots at optimum atmospheric temperature. Shoot materials (growing zones and leaves) were harvested at five points over a time course of four days, including a two‐day recovery period. Proteins were quantified by tandem mass tags and triple stage MS, using a method developed to overcome ratio compression in isobaric‐labelled quantitation. Over 3000 proteins in each of the tissues were quantified by multiple peptides. Proteins significantly differentially expressed as compared with the control included abscisic acid‐responsive and drought‐associated proteins. The data also contained evidence of a possible induction of a sugar signalling pathway.

Performance of broiler chickens offered nutritionally-equivalent diets based on two red grain sorghums with quantified kafirin concentrations as intact pellets or re-ground mash following steam-pelleting at 65 or 97°C conditioning temperatures

The Liverpool Plains is a fertile agricultural region in New South Wales, Australia. Two sorghums from the 2009 Liverpool Plains harvest, sorghums #3 and #5, were extensively characterised which included concentrations of kafirin and phenolic compounds plus rapid visco-analysis (RVA) starch pasting profiles. Diets based on these two sorghums were formulated to be iso-nitrogenous and iso-energetic and were offered to male Ross 308 broiler chicks from 7 to 28 days post--hatch as either intact pellets or reground mash following steam-pelleting at conditioning temperatures of either 65 or 97°C. Thus the feeding study consisted of a 2 × 2 × 2 factorial array of dietary treatments: two sorghum varieties, two feed forms and two conditioning temperatures. Each of the eight treatments was replicated six times with six birds per replicate cage. Assessed parameters included growth performance, nutrient utilisation, apparent starch and protein (N) digestibility coefficients and disappearance rates from the distal jejunum and distal ileum. Intact pellets supported higher (P < 0.001) feed intakes and weight gains by 9.83 and 9.08%, respectively, than reground mash diets. Feed conversion ratios of broilers offered diets steam-conditioned at 97°C were 2.46% inferior (P < 0.001) in comparison to 65°C diets and both apparent metabolizable energy (AME) and N-corrected AME (AMEn) were compromised. Broilers offered sorghum #3-based diets significantly (P < 0.001) outperformed their sorghum #5 counterparts in terms of weight gain by 3.75% (1,334 versus 1,223 g/bird), FCR by 4.81% (1.524 versus 1.601), AME by 1.06 MJ (13.61 versus 12.55 MJ/kg), ME:GE ratio (ME:GE) by 4.81% (0.806 versus 0.769) and AMEn by 1.03 MJ (12.38 versus 11.35 MJ/kg). The inferiority of sorghum #5 appeared to be associated with higher concentrations of kafirin (61.5 versus 50.7 g/kg) and conjugated phenolic acids, including ferulic acid (31.1 versus 25.6 µg/g). There were no significant differences in jejunal and ileal starch and protein (N) digestibility coefficients between the two sorghums. However, starch to protein (N) disappearance rate ratios from the distal jejunum were significantly (P < 0.001) correlated with ME:GE and AME. The multiple linear regression equations indicated that energy utilisation was enhanced by coupling rapidly digestible protein with slowly digestible starch, which suggests that bilateral bioavailability of starch and protein is pivotal to efficient energy utilisation.

Analysis of rice proteins using SDS-PAGE shotgun proteomics

In this chapter we describe the workflow used in our laboratory to analyze rice leaf samples using label-free shotgun proteomics based on SDS-PAGE fractionation of proteins. Rice proteomics has benefitted substantially from successful execution of shotgun proteomics techniques. We describe steps on how to proceed starting from rice protein extraction, SDS-PAGE, in-gel protein digestion with trypsin, nanoLC-MS/MS, and database searching using the GPM. Data from these experiments can be used for spectral counting, where simultaneous quantitation of several thousand proteins can be obtained.