Urine Samples [47, 48]
Urine is produced in the kidneys as a result of the ultrafiltration of
the plasma to eliminate waste products, such as urea and metabolites. Protein concentration in urine under physiological conditions
is very low, usually between 0 and 0.2 mg/mL [49].
Despite this, urine can be collected in large quantities and
noninvasively. Therefore, in spite of the low protein concentration,
adequate amounts of proteins can be obtained for proteomics
analysis. Moreover, urine has the advantage of making it possible
to obtain samples repeatedly, even over long periods of time.
Urine contains proteins originating from the blood and
kidneys, making this sample a good source for analysis of different
diseases [29].
All these characteristics make urine one of the most attractive
body fluids in clinical proteomics [50–52], although the variability
in protein concentration is a hindrance to the analysis of samples.
Urine Sample Preparation
[53, 54] (Fig. 6)
First, the urine can be centrifuged to eliminate the debris and tiny
stones present in this body fluid.
1. Samples are centrifuged at 14,000 Â g for 20 min, and the
precipitates are removed.
2. The supernatants are concentrated through an Amicon
® Ultra
3K device at 14,000 Â g for 30 min.
3. The upper layer is then collected and loaded in a gel (see next
section) before an in-gel digestion.
As urine samples have major proteins, in order to improve the
protein identification/quantification, it is possible to use protein
removal/enrichment techniques such as DTT depletion or nanoprotein treatment (see next sections for the removal of major
protein removal).
Blood, Serum, or Plasma
Samples
Human plasma (or serum) is one of the most useful samples for
protein biomarker discovery as it can easily be collected and contain
thousands of proteins, including those secreted or leaked into the
blood by normal cell and tissue processes or cell damage. The
protein concentration in these samples is estimated to 60–80 ng/
mL.
Therefore, in these samples, the presence, absence, or change in
expression of proteins might potentially indicate the presence or
progression of most disease states.
Sample Collection [55, 56]
Appropriate blood sample handling is necessary and crucial for
blood biomarker search by proteomics investigation [55].
l
To obtain plasma, blood samples are collected from the cubital
vein into blood collection tubes containing K2 ethylenediaminetetraacetic acid (EDTA) as an anticoagulant [55]. Plasma is
118
Maria del Pilar Chantada-Va ´ zquez et al.
Urine is produced in the kidneys as a result of the ultrafiltration of
the plasma to eliminate waste products, such as urea and metabolites. Protein concentration in urine under physiological conditions
is very low, usually between 0 and 0.2 mg/mL [49].
Despite this, urine can be collected in large quantities and
noninvasively. Therefore, in spite of the low protein concentration,
adequate amounts of proteins can be obtained for proteomics
analysis. Moreover, urine has the advantage of making it possible
to obtain samples repeatedly, even over long periods of time.
Urine contains proteins originating from the blood and
kidneys, making this sample a good source for analysis of different
diseases [29].
All these characteristics make urine one of the most attractive
body fluids in clinical proteomics [50–52], although the variability
in protein concentration is a hindrance to the analysis of samples.
Urine Sample Preparation
[53, 54] (Fig. 6)
First, the urine can be centrifuged to eliminate the debris and tiny
stones present in this body fluid.
1. Samples are centrifuged at 14,000 Â g for 20 min, and the
precipitates are removed.
2. The supernatants are concentrated through an Amicon
® Ultra
3K device at 14,000 Â g for 30 min.
3. The upper layer is then collected and loaded in a gel (see next
section) before an in-gel digestion.
As urine samples have major proteins, in order to improve the
protein identification/quantification, it is possible to use protein
removal/enrichment techniques such as DTT depletion or nanoprotein treatment (see next sections for the removal of major
protein removal).
Blood, Serum, or Plasma
Samples
Human plasma (or serum) is one of the most useful samples for
protein biomarker discovery as it can easily be collected and contain
thousands of proteins, including those secreted or leaked into the
blood by normal cell and tissue processes or cell damage. The
protein concentration in these samples is estimated to 60–80 ng/
mL.
Therefore, in these samples, the presence, absence, or change in
expression of proteins might potentially indicate the presence or
progression of most disease states.
Sample Collection [55, 56]
Appropriate blood sample handling is necessary and crucial for
blood biomarker search by proteomics investigation [55].
l
To obtain plasma, blood samples are collected from the cubital
vein into blood collection tubes containing K2 ethylenediaminetetraacetic acid (EDTA) as an anticoagulant [55]. Plasma is
118
Maria del Pilar Chantada-Va ´ zquez et al.