Targeted serum proteomics of longitudinal samples from newly diagnosed youth with type 1 diabetes distinguishes markers of disease and C-peptide trajectory

Aims/hypothesis There is a growing need for markers that could help indicate the decline in beta cell function and recognise the need and efficacy of intervention in type 1 diabetes. Measurements of suitably selected serum markers could potentially provide a non-invasive and easily applicable solution to this challenge. Accordingly, we evaluated a broad panel of proteins previously associated with type 1 diabetes in serum from newly diagnosed individuals during the first year from diagnosis. To uncover associations with beta cell function, comparisons were made between these targeted proteomics measurements and changes in fasting C-peptide levels. To further distinguish proteins linked with the disease status, comparisons were made with measurements of the protein targets in age- and sex-matched autoantibody-negative unaffected family members (UFMs). Methods Selected reaction monitoring (SRM) mass spectrometry analyses of serum, targeting 85 type 1 diabetes-associated proteins, were made. Sera from individuals diagnosed under 18 years (n=86) were drawn within 6 weeks of diagnosis and at 3, 6 and 12 months afterwards (288 samples in total). The SRM data were compared with fasting C-peptide/glucose data, which was interpreted as a measure of beta cell function. The protein data were further compared with cross-sectional SRM measurements from UFMs (n=194). Results Eleven proteins had statistically significant associations with fasting C-peptide/glucose. Of these, apolipoprotein L1 and glutathione peroxidase 3 (GPX3) displayed the strongest positive and inverse associations, respectively. Changes in GPX3 levels during the first year after diagnosis indicated future fasting C-peptide/glucose levels. In addition, differences in the levels of 13 proteins were observed between the individuals with type 1 diabetes and the matched UFMs. These included GPX3, transthyretin, prothrombin, apolipoprotein C1 and members of the IGF family. Conclusions/interpretation The association of several targeted proteins with fasting C-peptide/glucose levels in the first year after diagnosis suggests their connection with the underlying changes accompanying alterations in beta cell function in type 1 diabetes. Moreover, the direction of change in GPX3 during the first year was indicative of subsequent fasting C-peptide/glucose levels, and supports further investigation of this and other serum protein measurements in future studies of beta cell function in type 1 diabetes. Graphical Abstract Supplementary Information The online version contains peer-reviewed but unedited supplementary material available at 10.1007/s00125-023-05974-9.


Protein Selection: Previous Type 1 Diabetes Proteomics studies
Metz et al. used high-resolution capillary LC-MS/MS and intensity based quantification for the analysis of immuno-affinity depleted sera, reporting the first serum proteomics comparison of recently diagnosed T1D patients and matched controls (n=10 vs 10).They later followed this up in a second discovery study, using similar analytical methodology with pooled sera (n=10 vs 10) and a larger scale targeted mass spectrometry-based validation study (T1D=50, control =100, T2D = 50) 1,3 .
From the panel of detected markers, they concluded that T1D caused dysregulation of innate immunity.From their validations they reported that peptides from platelet basic protein and C1 inhibitor (SEPRING1) achieved excellent discrimination between T1D and controls.
Zhi et al. used a spectral counting proteomics approach to quantify serum markers, comparing three pools of immuno-affinity depleted sera, each from 10 T1D patients with three similar controls pools 2 .
From their panel of 21 candidate markers, immuno-assay validation was made for six targets in an extended cohort (1139 T1D and 849 Aab-).The latter targets included adiponectin, insulin-like growth factor binding protein 2, C-reactive protein, serum amyloid protein A, transforming growth factor beta induced and myeloperoxidase.
Oliveira et al. 7 used data-independent, label-free mass spectrometric analysis of immuno-affinity depleted sera to compare patients and control subjects (n=30 vs 30), reporting eight markers with overlap of some of the markers from the latter two studies.
T1D related proteomics studies have also included the analysis of sera from children who tested positive for T1D associated autoantibodies 6 , temporal analysis of at-risk healthy controls and analysis of the changes leading to T1D onset 5 .In our previous investigations, we have used both isobaric labelling and label free methods to measured serum proteomes of children from a HLA-conferred T1D risk group to identify changes associated with maturation, the appearance of autoantibodies and diagnosis (n=19, vs n=19) 4,11 .
In the study of islet autoantibody-positive children, von Toerne et al. 6 employed label free analysis of immuno-affinity depleted sera for discovery and targeted mass spectrometry-based validation.
Here, they reported peptide signatures indicative islet autoimmunity could be found prior to the diagnosis of type 1 diabetes.
Liu and co-workers 5 used isobaric labelling of depleted sera to compare sera from children developing type 1 diabetes with age matched controls.They found statistically significant differences for 13 proteins, including catalase and superoxide dismutase, which were validated by ELISA.Taken together, they reported that oxidative stress related proteins were dysregulated before islet autoimmunity.Markers reported in the above studies and selected for measurement are indicated in the following tables.

ESM Table 1: Proteins measured by Targeted Mass Spectrometry
Targets were selected from a range of studies of type 1 diabetes and other serum/plasma proteomics investigations.For presentation, these are subdivided into general groups, with the control/reference proteins either in the control group or marked with †.The peptides measured are indicated in ESM Table 2 a) Targets at a glance.For quick reference, the selected proteins are tabulated with their gene name and group assignments.Malmström R, Diabetes.1998; 13 Bereket A, et al.J Clin Endocrinol Metab.1995; 14 Peet A, Eur J Endocrinol. 2015; 15 Jilma B, Thromb Haemost.1996; 16   As an early project milestone, detailed analysis of blood samples collected from first 100 newly diagnosed (ND) individuals have been made.These multi-omics analyses, targeting microRNA, mRNA, metabolites, lipids, proteins, and immune cell populations, together with data on C -peptide levels, fasting glucose and mixed meal tolerance tests, present a baseline for reference in the newly diagnosed, and are to be described elsewhere.

Apolipoprotein
The samples analysed in this proteomics study were collected between 2018 and 2019 from the first 100 subject newly diagnosed with type 1 diabetes.With this consecutive recruitment approach, subjects between the ages of one to 45 years were included on the basis of an even gender distribution, biosample availability and positivity for at least one diabetes-related autoantibody (GADA, IA-2A, ZnT8A).To reduce the influence of age in these targeted proteomics comparisons, only individuals diagnosed up to the age of 18 were considered, together with age and sex matched unaffected members of type 1 diabetes families.The data from the newly diagnosed were compared with Cpeptide and fasting glucose measurements (used as a ratio of C-peptide/glucose).C-peptide data was collected up until 2020.The samples from the newly diagnosed included blood/sera drawn within 6 weeks of diagnosis, then 3 months, 6 months and up to one year afterwards.

INNODIA
Project Overview: https://www.innodia.eu/The INNODIA project (Innovative approaches to understanding and arresting type 1 diabetes) was established in 2015 as a pan-European consortium with the principle aim to fight T1D.Participating clinical centres in thirteen countries have monitored newly diagnosed individuals and unaffected family members, collecting biological samples to define the natural history of the disease.Subsequent actions in the study have included the launch of clinical trials with immunosuppressive drugs to prolong beta cell function (https://www.innodia.eu/harvest/).