3 Methods
3.1 Methods for
Protein Extraction
3.1.1 Protein Extractions
from Different Body Fluids
[27, 28] (See Note 4)
In the last years, there is an increasing demand for non- or minimally invasive methods in medical diagnosis and prognosis. The
body fluids that are constantly produced can be collected by noninvasive means such as tears, saliva, and urine. These body fluids
(e.g., blood, urine, tear, and saliva) contain a lot of proteins that are
secreted by different tissues and organs in the body. Changes in
their proteomes can reflect the state of the body and can indicate a
disease state. Moreover, the analysis of protein changes in these
body fluids is minimally invasive, making it very attractive and
useful for clinical diagnostics. Although samples such as serum or
plasma are mainly useful, these samples are complicated and highly
challenging due to the large dynamic range of protein abundance
that can complicate the low-abundance protein identification.
Therefore, the concentration range goes over at least 10 orders of
magnitude more, from the most abundant albumin (~50 mg/mL)
to cytokines of low abundance (e.g., 4.2, 7.4, and 11.2 pg/mL for
interleukin-6, interleukin-17, and TNF-α, respectively) in normal
individuals. More recently, there have been several studies of other
body fluids such as the urinary proteome or tear proteome that
have a less dynamic range of protein abundance, making its analysis
easier [29].
As a summary, although it is also true that in some cases it is
impossible to avoid the analysis of body fluids collected by invasive
techniques and considering the possibility of infections and complications and costs of medical interventions required for the collection of these body fluids (as cerebrospinal fluid), it is also
necessary to search for noninvasively collectable body fluids that
can give us good values in disease diagnosis.
Here, we present several protocols to make a SWATH-MS
quantification in some body fluids. It is necessary to indicate that
the presence of highly abundant proteins, such as albumin in
serum, α-amylase in saliva, lactotransferrin and lysozyme-C in
tears, and the low amount of available sample, may require the
utilization of protein depletion and/or enrichment methods previous to the protein analysis.
Saliva Samples [28, 29]
Saliva is composed of more than 99% water and is a complex
mixture secreted from major and minor salivary glands and the
gingival crevice [30]. Therefore, the major part of saliva is composed of some electrolytes, proteins, mucins, and nitrogenous
products such as urea [31]. It is known that saliva can contain
more than 2000 proteins, the most abundant of which are
α-amylase mucins, proline-rich peptides, cystatins, and serum albumin [32]. Although some authors have shown high variability in
protein content depending on collection time, gender, age, and
114
Maria del Pilar Chantada-Va ´ zquez et al.
3.1 Methods for
Protein Extraction
3.1.1 Protein Extractions
from Different Body Fluids
[27, 28] (See Note 4)
In the last years, there is an increasing demand for non- or minimally invasive methods in medical diagnosis and prognosis. The
body fluids that are constantly produced can be collected by noninvasive means such as tears, saliva, and urine. These body fluids
(e.g., blood, urine, tear, and saliva) contain a lot of proteins that are
secreted by different tissues and organs in the body. Changes in
their proteomes can reflect the state of the body and can indicate a
disease state. Moreover, the analysis of protein changes in these
body fluids is minimally invasive, making it very attractive and
useful for clinical diagnostics. Although samples such as serum or
plasma are mainly useful, these samples are complicated and highly
challenging due to the large dynamic range of protein abundance
that can complicate the low-abundance protein identification.
Therefore, the concentration range goes over at least 10 orders of
magnitude more, from the most abundant albumin (~50 mg/mL)
to cytokines of low abundance (e.g., 4.2, 7.4, and 11.2 pg/mL for
interleukin-6, interleukin-17, and TNF-α, respectively) in normal
individuals. More recently, there have been several studies of other
body fluids such as the urinary proteome or tear proteome that
have a less dynamic range of protein abundance, making its analysis
easier [29].
As a summary, although it is also true that in some cases it is
impossible to avoid the analysis of body fluids collected by invasive
techniques and considering the possibility of infections and complications and costs of medical interventions required for the collection of these body fluids (as cerebrospinal fluid), it is also
necessary to search for noninvasively collectable body fluids that
can give us good values in disease diagnosis.
Here, we present several protocols to make a SWATH-MS
quantification in some body fluids. It is necessary to indicate that
the presence of highly abundant proteins, such as albumin in
serum, α-amylase in saliva, lactotransferrin and lysozyme-C in
tears, and the low amount of available sample, may require the
utilization of protein depletion and/or enrichment methods previous to the protein analysis.
Saliva Samples [28, 29]
Saliva is composed of more than 99% water and is a complex
mixture secreted from major and minor salivary glands and the
gingival crevice [30]. Therefore, the major part of saliva is composed of some electrolytes, proteins, mucins, and nitrogenous
products such as urea [31]. It is known that saliva can contain
more than 2000 proteins, the most abundant of which are
α-amylase mucins, proline-rich peptides, cystatins, and serum albumin [32]. Although some authors have shown high variability in
protein content depending on collection time, gender, age, and
114
Maria del Pilar Chantada-Va ´ zquez et al.