Heavy breathing proteins
Chronic obstructive pulmonary disease (COPD) is one of the most common adult respiratory diseases worldwide and the fourth leading cause of death. The American Lung Association estimates that about 15 million US citizens are affected. In the UK, the 30,000 deaths annually mean that more die of COPD than from breast, colon or prostate cancer. Despite the high prevalence, COPD is preventable and treatable, early diagnosis and treatment slowing its progression.
This disease is, in reality, a set of multiple conditions that includes emphysema and chronic bronchitis. Sufferers experience coughing and breathlessness. COPD is usually caused by smoking and for this reason it is often considered as a self-inflicted disease. "Stop smoking and the symptoms will abate." That is true to some extent but for the elderly and the severely affected it can be too late to have any real effect.
In emphysema, the walls between the tiny air sacs in the lungs are destroyed, leading to a smaller number of large sacs that are unable to exchange oxygen and carbon dioxide efficiently. As a result, insufficient oxygen circulates around the body. With chronic bronchitis, the airways in the lungs become inflamed and thickened and excessive mucus is produced, contributing to coughing and breathing difficulties.
Although much is known about COPD, the molecular basis of the disease and the mechanisms that govern its initiation and progression are poorly understood. In a new approach, researchers in Italy and the USA have assessed the suitability of proteomics methods to try and identify changes in protein expression that are related to the disease pathophysiology. They analysed sputum from people affected by COPD to different degrees and compared the protein profiles to pick out potential biomarkers.
The sputum was collected from 56 subjects by a standardised method using saline induction. They were categorised into 5 groups: healthy lifelong non-smokers with no airway disease (group A); healthy smokers (sic) with no airflow obstruction or mucus hypersecretion (B); smokers without airflow obstruction but complaining about cough and phlegm (C); smokers with airflow obstruction but no emphysema (D); the COPD group with airflow obstruction plus emphysema (E). The classification was deemed necessary in order to "anticipate the probable proteomic content of induced sputum for each clinical phenotype and for linking these proteomic differences to pathophysiological differences between the COPD groups."
The sputum was treated with dithiothreitol and centrifuged, the supernatant being mixed with chloroform-methanol. The proteins collecting at the aqueous interface were extracted, reduced, alkylated and digested with trypsin. All digests were analysed by HPLC-tandem mass spectrometry with electrospray ionisation and the peptides were matched to proteins using standard MS/MS software.
A total of 203 proteins were identified, probably representing the most abundant proteins in sputum. Most were matched with good certainty, using at least three peptide sequences. For identifications based on one or two sequences, the tandem mass spectra were examined visually for confirmation.
Many proteins had been identified previously in bronchoalveolar lavage fluid (BALF) but 118 were detected for the first time that had not been found in earlier studies of sputum or BALF using two-dimensional gel electrophoresis (2-DE). They included proteins with high isoelectric points (up to 12) which are difficult to detect on 2-DE gels, as well as 21 proteins with molecular masses greater than 150 kDa.
The novel proteins included large mucins, low-molecular-mass immune proteins and highly cationic eosinophils. Moreover, many immunoglobulins were found that had previously been grouped generically as immunoglobulin G and A, but were identified now with specific heavy and light chain compositions.
As they had anticipated, the researchers found that several proteins were detected more frequently in some groups of subjects than others. For instance, Zn-alpha2-glycoprotein was found in 71% of group A samples but in less than 10% of all the other groups. This protein is known to be down-regulated in patients with pulmonary injury.
The protein known as Clara cells appeared in 100% of group A but in lesser proportions of the other subjects. It has been suggested that Clara cells have an anti-inflammatory function induced by cigarette smoke, so loss of this protective effect might contribute to the progression of COPD.
Other proteins were found at greater frequencies in the diseased subjects. Histone H4 was found only in group E, the most severely affected patients. Its presence indicates that nuclear acetylation might be disrupted in COPD and emphysema. Similarly, cathepsin G was found only in group E, at a frequency of 27% and mucin5A/C was found at a level of 43% in group A but at higher levels in all of the other groups.
In total, 14 proteins displayed differential expression across the five disease groups. Closer examination of their functions will give clinicians a clearer understanding of the disease while a broader study of their frequency could help to establish a panel of proteins as biomarkers for the different stages of COPD.
Article by Steve Down
Source:
Proteomics and Genomics
This disease is, in reality, a set of multiple conditions that includes emphysema and chronic bronchitis. Sufferers experience coughing and breathlessness. COPD is usually caused by smoking and for this reason it is often considered as a self-inflicted disease. "Stop smoking and the symptoms will abate." That is true to some extent but for the elderly and the severely affected it can be too late to have any real effect.
In emphysema, the walls between the tiny air sacs in the lungs are destroyed, leading to a smaller number of large sacs that are unable to exchange oxygen and carbon dioxide efficiently. As a result, insufficient oxygen circulates around the body. With chronic bronchitis, the airways in the lungs become inflamed and thickened and excessive mucus is produced, contributing to coughing and breathing difficulties.
Although much is known about COPD, the molecular basis of the disease and the mechanisms that govern its initiation and progression are poorly understood. In a new approach, researchers in Italy and the USA have assessed the suitability of proteomics methods to try and identify changes in protein expression that are related to the disease pathophysiology. They analysed sputum from people affected by COPD to different degrees and compared the protein profiles to pick out potential biomarkers.
The sputum was collected from 56 subjects by a standardised method using saline induction. They were categorised into 5 groups: healthy lifelong non-smokers with no airway disease (group A); healthy smokers (sic) with no airflow obstruction or mucus hypersecretion (B); smokers without airflow obstruction but complaining about cough and phlegm (C); smokers with airflow obstruction but no emphysema (D); the COPD group with airflow obstruction plus emphysema (E). The classification was deemed necessary in order to "anticipate the probable proteomic content of induced sputum for each clinical phenotype and for linking these proteomic differences to pathophysiological differences between the COPD groups."
The sputum was treated with dithiothreitol and centrifuged, the supernatant being mixed with chloroform-methanol. The proteins collecting at the aqueous interface were extracted, reduced, alkylated and digested with trypsin. All digests were analysed by HPLC-tandem mass spectrometry with electrospray ionisation and the peptides were matched to proteins using standard MS/MS software.
A total of 203 proteins were identified, probably representing the most abundant proteins in sputum. Most were matched with good certainty, using at least three peptide sequences. For identifications based on one or two sequences, the tandem mass spectra were examined visually for confirmation.
Many proteins had been identified previously in bronchoalveolar lavage fluid (BALF) but 118 were detected for the first time that had not been found in earlier studies of sputum or BALF using two-dimensional gel electrophoresis (2-DE). They included proteins with high isoelectric points (up to 12) which are difficult to detect on 2-DE gels, as well as 21 proteins with molecular masses greater than 150 kDa.
The novel proteins included large mucins, low-molecular-mass immune proteins and highly cationic eosinophils. Moreover, many immunoglobulins were found that had previously been grouped generically as immunoglobulin G and A, but were identified now with specific heavy and light chain compositions.
As they had anticipated, the researchers found that several proteins were detected more frequently in some groups of subjects than others. For instance, Zn-alpha2-glycoprotein was found in 71% of group A samples but in less than 10% of all the other groups. This protein is known to be down-regulated in patients with pulmonary injury.
The protein known as Clara cells appeared in 100% of group A but in lesser proportions of the other subjects. It has been suggested that Clara cells have an anti-inflammatory function induced by cigarette smoke, so loss of this protective effect might contribute to the progression of COPD.
Other proteins were found at greater frequencies in the diseased subjects. Histone H4 was found only in group E, the most severely affected patients. Its presence indicates that nuclear acetylation might be disrupted in COPD and emphysema. Similarly, cathepsin G was found only in group E, at a frequency of 27% and mucin5A/C was found at a level of 43% in group A but at higher levels in all of the other groups.
In total, 14 proteins displayed differential expression across the five disease groups. Closer examination of their functions will give clinicians a clearer understanding of the disease while a broader study of their frequency could help to establish a panel of proteins as biomarkers for the different stages of COPD.
Article by Steve Down
Source:
Proteomics and Genomics
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