SARS-CoV-2 infection leads to distinct changes in proteomic and peptidomic profiles and posttranslational modifications

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) causes the highly contagious coronavirus disease-19 (COVID-19). COVID-19 has evolved into a pandemic affecting lives and economies worldwide.

COVID-19 mainly results in mild to severe respiratory symptoms and may result in other complications such as lung inflammation and damage, thrombosis, stroke, renal failure, neurological disorders, etc. Currently, a complete understanding of COVID-19 disease pathology and the short-term and long-term effects of the disease does not exist.

Study: Protein Posttranslational Signatures Identified in COVID-19 Patient Plasma. Image Credit: anon_tae/Shutterstock

A recent study released as a pre-print on the bioRxiv* server attempts to understand the protein-level alterations in COVID-19 patients as a result of SARS-CoV-2 infection and additionally aims to identify the distinct proteomic and posttranslational signatures pertaining to COVID-19. The scientists in the present study performed proteomic analysis of plasma samples from hospitalized COVID-19 patients with severe symptoms and compared these with normal healthy controls.

Background

The scientists in the present study compared the plasmas from six COVID-19 patients with normal healthy controls to detect changes in proteins and peptides in their plasma peptidomes due to COVID-19 infection.

The plasma peptides from the collected samples were isolated using size exclusion under denaturing conditions followed by C18 reverse phase clean up and subsequently, LC-MS/MS was performed on them without subjecting them to proteolysis treatment.

On comparing the peptides identified in the plasma samples it was found that the number of peptides identified in the COVID-19 samples was less when compared to the control samples. Notably, this finding was not in alignment with the fact that high levels of proteolysis were observed in SARS-CoV-2 infection.  

COVID-19 related changes in plasma peptidomes were further analyzed by segregating the identified peptides based on their parent proteins. Plots were made for the combined intensities of the peptides that were changed in the case of each protein and further plots were made for the combined intensities grouped by proteins.

It was found that combined peptide intensities for each protein were higher in the case of controls when compared to COVID-19 patient samples. This is in correlation with the finding that the number of peptides identified was higher in the control samples.

Notably, peptides of certain groups of proteins such as Alpha-1-antitrypsin, C4a anaphylatoxin, and Serum amyloid A-2 were significantly increased in the case of COVID-19 patient plasma. Interestingly these proteins have been associated with COVID-19 pathology. Alpha-1-antitrypsin is a protease inhibitor that plays a role in the regulation of plasma proteolysis, C4 anaphylatoxin, and Serum amyloid A-2 play a role in the immune response. These processes have been implicated in COVID-19 disease. The increased levels of peptides of these proteins in COVID-19 patient samples may indicate a direct relation between the proteolysis of these proteins and COVID-19 pathology.  

The peptides that were present in higher levels in control samples belonged to proteins that were normally proteolyzed during routine functioning of the body. They were observed to be less in COVID-19 samples as the levels of these proteins may have decreased as a result of infection.

Alpha-fibrinogen is a blood-clotting protein and serglycin plays a role in the biology of blood cells. Significantly changed individual peptides of these proteins were identified that were abundant in the case of COVID-19 patient samples when compared to the control. This indicates that these two proteins undergo proteolytic changes in response to SARS-CoV-2 infection that is distinct from that observed in normal physiology. These proteins may serve as biomarkers for investigating COVID-19.

Global proteome changes

The scientists further analyzed the plasma samples employing shotgun proteomics and observed that the levels of twelve proteins were significantly increased while that of 35 proteins were decreased in COVID-19 patient samples when compared to controls.

iBAQ is calculated as the sum of all the peptide intensities divided by the number of observable peptides of a protein and it was employed in the present study to evaluate the relative abundance of proteins.

iBAQ assessments revealed that the levels of serum amyloid A-2, serum amyloid A-1 and C-reactive protein were elevated in COVID-19 patient plasma. Serum amyloid A-1 and C-reactive protein levels are known to be elevated in response to inflammation and therefore may be increased in response to SARS-CoV-2 infection.

Notably, it was found that the proteins that were found to be decreased in COVID-19 patient samples were those responsible for normal physiological functions in the body. These include proteins involved in anti-inflammatory response such as apolipoprotein A-IV and C-III, overall protective functions such as serum paraoxonase, hormone and vitamin transport proteins such as retinol-binding protein, and transthyretin. The decreased levels of these proteins in COVID-19 patients may be the effect of SARS-CoV-2 infection.

The findings from this study suggest that when compared to healthy controls, samples from COVID-19 patients exhibit distinctly altered levels of proteins involved in inflammation, immune response, and normal homeostasis which may be a consequence of SARS-CoV-2 infection.

Does a distinct posttranslational COVID-19 code exist?

In order to identify the altered posttranslational modifications (PTMs) as a result of COVID-19 infections, a software package called pFIND was employed which can identify PTMs and chemical modifications with the help of mass shifts.

Statistically significant differences were observed between COVID-19 and control in the case of 82 PTMs. Most of these PTMs were identified to occur in response to drugs and environmental factors and not under normal physiological conditions. The PTMs that were known to occur naturally under physiological conditions alone were selected for further analysis.

After excluding the PTMs that were not naturally induced it was found that only a few PTMs exhibited significant differences between COVID-19 patient and control samples.

In COVID-19 patients, phosphorylation on threonine (Thr) was found to be increased around two fold and side chain arginylation of Asp and Glu residues decreased 1.5 fold. In the case of controls arginine (Arg) deamidation increased around two fold.

Further investigations were performed on the PTMs, arginylation, and Thr phosphorylation as these have been earlier reported to have a global regulatory role. The scientists found that in COVID-19 patient samples, in addition to the changes in the levels of these PTMs the repertoire of arginylation and Thr phosphorylation sites were also altered when compared to the controls. This finding indicates that in COVID-19 patients, the target proteins bearing these altered PTMs may exhibit distinct behavior as a result of SARS-CoV-2 infection. This may impact functions that may play a role in COVID-19 infection such as blood coagulation and immune response.

Further investigations are needed to explore if arginylation/phosphorylation on the altered PTM sites may play role in COVID-19 disease progression.

Additionally, levels of Glycosylation which is N-linked are higher in controls, and levels of glycosylation that is S-linked and present at N-terminals are higher in COVID-19 patient samples.

Further, differences in amino acid substitutions have been observed between control and COVID-19 patient samples especially the glutamic acid (Glu) to aspartic acid (Asp) substitutions. These single nucleotide polymorphisms (SNPs) may not be the result of SARS-CoV-2 infection, however, they may indicate the genetic modifications that may increase the risk of developing COVID-19.

Conclusion

The present study has attempted for the first time to analyze and compare the global proteome and peptidome of plasma samples from COVID-19 patients with healthy controls. The study has identified certain proteins associated with inflammation and immune response whose levels are elevated in COVID-19. A few of these proteins including fibrinogen undergo proteolytic events that are different when compared to normal controls. Additionally, distinct posttranslational signatures were also observed in COVID-19 patient samples compared to controls. There were altered levels of glycosylation, citrullination, Thr phosphorylation, and arginylation.

The findings from this study indicate that as a result of SARS-CoV-2 infection there occurs comprehensive changes in protein processing and regulation. There also exists a possibility of a  distinct posttranslational COVID-19 code. The protein-level changes observed in this study may be related to the poorly understood symptoms associated with COVID-19. Further investigations in this area will help enhance our understanding of COVID-19 disease progression and improve the diagnostics and treatments available.

*Important notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behaviour, or treated as established information.

Written by

Dr. Maheswari Rajasekaran

Maheswari started her science career with an undergraduate degree in Pharmacy and later went on to complete a master’s degree in Biotechnology in India. She then pursued a Ph.D. at the University of Arkansas for Medical Sciences in the USA.