Changes in meat quality traits and sarcoplasmic proteins during aging in three different cattle breeds R. Marino, M. Albenzio, A. della Malva, M. Caroprese, A. Santillo, A. Sevi PII: DOI: Reference:
S0309-1740(14)00155-7 doi: 10.1016/j.meatsci.2014.05.024 MESC 6431
To appear in:
Meat Science
Received date: Revised date: Accepted date:
30 July 2013 27 February 2014 30 May 2014
Please cite this article as: Marino, R., Albenzio, M., della Malva, A., Caroprese, M., Santillo, A. & Sevi, A., Changes in meat quality traits and sarcoplasmic proteins during aging in three different cattle breeds, Meat Science (2014), doi: 10.1016/j.meatsci.2014.05.024
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Changes in meat quality traits and sarcoplasmic proteins
SC R
IP
T
during aging in three different cattle breeds
MA
NU
R. Marino*, M. Albenzio, A. della Malva, M. Caroprese, A. Santillo, A. Sevi
Dipartimento di Scienze Agrarie, degli Alimenti e dell’Ambiente, University of Foggia, Via
TE
D
Napoli, 25, 71121, Foggia, Italy.
*Corresponding author: Tel.: +39 0881 589330; fax: +39 0881589502.
AC
CE P
Email address: [email protected] (R.Marino)
ACCEPTED MANUSCRIPT Abstract The effects of breed and aging time (1, 7, 14, 21d) were evaluated on physical meat properties and
T
on sarcoplasmic proteins changes in 24 young bulls from Romagnola x Podolian, Podolian and
IP
Friesian breed. Aging affects lightness showing an increase in all breeds while changes in redness
SC R
varied according to the breed. Podolian breed showed meat with the darkest and the reddest color and the lowest drip loss compared to the other breeds. Extending aging to 21d reduced drip loss
NU
from meat. SDS-PAGE and 2DE showed that many changes in the sarcoplasmic proteins occurred among breeds and during aging. During post-mortem some sarcoplasmic proteins decline in
MA
intensity after 21d highlighting that they were susceptible to aging. Proteins identification and western blotting showed the presence of myosin light chains, Troponin T and tropomyosin proteins
D
during aging, suggesting a degradation of myofibers and a more intense proteolysis especially in
CE P
TE
Podolian breed.
AC
Keywords: breed, aging, water holding capacity, sarcoplasmic proteins, proteolysis.
1. Introduction Eating satisfaction is one of the most important characteristics by which consumers judge meat quality after purchase (Grunert, Bredahl, & Brunso, 2004); tenderness, color and juiciness are the major components of beef palatability (Serra, Ruiz-Ramirez, Arnau, & Gou, 2005; Muchenje et al., 2009). Several studies (Ruiz de Huidobro, Miguel, Onega, & Blazquez, 2003; Revilla, & VivarQuintana, 2006) reported that these parameters are dependent on the rate and extent of post mortem aging. A number of factors contribute to post mortem tenderization of meat, in particular, many
ACCEPTED MANUSCRIPT authors (Koohmaraie, 1996; Taylor, Geesink, Thompson, Koohmaraie, & Goll, 1995; Kolczak, Pospiech, Palka, & Lacki, 2003; Huff-Lonergan, Zhang, & Lonergan, 2010) indicate that
T
proteolytic degradation of myofibrillar proteins plays an important role in tenderization showing
IP
ultrastructural changes in skeletal muscle.
SC R
Besides to myofibrillar proteins, sarcoplasmic fraction is the other compound of muscle proteins that account for 30-35% of total proteins and is primarily represented by glycolitic enzymes and
NU
myoglobin. Previous research (Ohlendieck, 2010) reported that the alteration of the sarcoplasmic fraction is not directly involved in the muscle tenderness, because the sarcoplasmic proteins have
MA
non structural functions and they are soluble in situ. Even though, studies in pig reported that the denaturation of sarcoplasmic proteins has an impact on meat quality parameters such as color and
D
water holding capacity (Sayd et al., 2006; Joo, Kauffman, Kim, & Park, 1999).
TE
Consumers’ choices are concerned not only about meat quality but also its origin (Grunert et al.,
CE P
2004). In particular, the use of rustic or autochthonous breed has the advantage that these animals are closely related to the environment and maintain the biodiversity (Revilla et al., 2006; Cuvelier et al., 2006). A large number of genetically distinct cattle breed are reared in Italy and this genetic
AC
diversity produces meat with many peculiar features. The effect of breed and aging on meat tenderness and on proteolytic pattern of myofibrillar proteins are reported in a previous study (Marino et al., 2013), data showed that meat tenderness and proteolysis were different among breeds with different purpose. To our knowledge, limited data are available on changes in meat sarcoplasmic proteins among different cattle breeds during aging. Therefore, in the present study we investigated the effect of breed and aging time on physical meat properties (pH, color, water holding capacity) and related sarcoplasmic proteins changes in longissimus dorsi muscle in three breeds with different productive purpose.
2. Materials and methods
ACCEPTED MANUSCRIPT 2.1. Animals and meat sampling Twenty four young bulls of three cattle breeds with different productive purpose, dairy (Friesian),
T
beef (Romagnola x Podolian crossbreed) and rustic (Podolian), were used in this study, details on
IP
breed characteristics are described in Marino et al. (2013). Animals (8 for each breed) were reared
SC R
intensively during the finishing period and were slaughtered at 19 months of age, according to industrial routines used in Italy and to the EU rule n. 119/1993.
NU
24 h post mortem Longissimus dorsi (LD) muscle was removed from each half of the carcass. Each muscle was divided longitudinally into two sections resulting in four samples for each animal.
MA
Then, each sample was stored at 2°C under vacuum packaging and analyzed at 1, 7, 14 and 21 d of aging, respectively. Cranial and caudal sections were randomized across aging time. pH, color
D
parameters, Warner Bratzler shear force (WBS), water holding capacity and changes in
TE
sarcoplasmic proteins were estimated. The effects of aging and breed on WBS are reported in a
CE P
previous paper (Marino et al., 2013).
AC
2.2. pH measurement, color measurements and water holding capacity (WHC) Ten g of each sample was blended with 90 ml of peptone water in a Stomacher bag for 30 s using a Stomacher (Interscience, France), the pH of the mixture was measured using a pH meter (Crison Instrument, Barcelona, Spain). Color was measured using a color meter Minolta CR 400 (Konica Minolta, Osaka, Japan) on 1 cm thick steaks after storage at 3 ± 1 °C for 1 h. Results were expressed as L* (lightness), a* (redness) and b* (yellowness) according to the standard conditions of the Commission International d’Eclairage (CIE). The values were measured in five location on the surface of each slice after 1, 7, 14 and 21 days.
ACCEPTED MANUSCRIPT Water holding capacity was measured as thawing loss. 1 cm thick frozen steak from each sample was placed on plastic netting over a polystyrene tray and stored in a plastic bag for 48 h at 4°C.
T
After storage, the meat was removed from the tray and the weight of the tray plus the juice was
SC R
thawing loss (%) = [(Wt + j − Wt)/ (Wi + m − Wt)] × 100,
IP
recorded. Thawing loss was expressed as a percentage of the initial weight of the meat:
where Wt is the weight of the empty tray, Wt + j is the weight of the tray plus the juice, and Wi + m
2.3. Extraction of sarcoplasmic proteins
MA
NU
is the weight of the tray with meat.
D
Samples were freed of connective and adipose tissue, after 2.5 g were homogenized with an Ultra
TE
Turrax homogenizer (IKA T18 basic, Germany) with 10 ml of 0.003M Phosphate Buffer at pH 7,
CE P
for 3 min. The homogenate was centrifuged under refrigeration at 4°C and 8000 x g (Eppendorf 5810 R, Eppendorf AG, Hamburg, Germany) for 20 min. After centrifugation the supernatant was aliquotated and frozen at -80°C. Protein concentrations were determined by the Bradford protein
AC
assay (Bio-Rad Laboratories, Hercules, CA) using bovine serum albumin (BSA) as a standard.
2.4 Gel sample preparation and SDS-PAGE Analysis Sarcoplasmic proteins were resolved by Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in a gradient gel 8-18%. The run was performed in a continuous buffer system using a Protean II xi vertical slab gel unit (Bio-Rad Laboratories, Hercules, CA). Gels were stained with Coomassie blue G250. Destained gel images were acquired by the Chemi Doc EQ system (Bio-Rad Laboratories, Hercules, CA) using a white light conversion screen and analyzed with the Quantity One software (Bio-Rad Laboratories, Hercules, CA) to determine the signal intensity (optical intensity) of the defined bands. Identification of the protein molecular weight was done by
ACCEPTED MANUSCRIPT comparison with a known molecular weight standard (precision plus protein standard-broad range, Bio-Rad, Laboratories, Hercules, CA). With the sum of the intensity of the defined bands in a lane
T
set a 100%, the relative quantity of each band was determined as the percentage of the signal
2.5. Two dimensional Gel Electrophoresis (2DE)
SC R
IP
intensity of the defined band in a lane.
NU
Protein separation in the first dimension was made in immobilized pH gradient (IPG) dry strips (17 cm IPG strips, Bio-Rad Laboratories, Hercules, CA), spanning the pH regions 3-10 for the
MA
sarcoplasmic extract using an IPG Protean IEF Cell (Bio-Rad Laboratories, Hercules, CA). The sarcoplasmic extract was suspended in isoelectrofocusing IPG sample buffer (ready-Prep
D
Rehydratation/Sample Buffer, Bio-Rad Laboratories, Hercules, CA). The IPG strips were
TE
equilibrated at room temperature for 15 min in equilibration buffers I and II (Bio-Rad Laboratories, Hercules, CA). Two dimensional separation was performed on a Protean II xi vertical slab gel unit
CE P
(Bio-Rad Laboratories, Hercules, CA) using SDS-PAGE gradient 8–18%. A broad range molecular weight electrophoresis calibration kit (Bio-Rad Laboratories, Hercules, CA) was used as standard.
AC
Gels were fixed and stained with Coomassie blue G250 (Bio-Rad Laboratories, Hercules, CA). The destained gels were acquired by the Chemi Doc EQ system (Bio-Rad Laboratories, Hercules, CA) using a white light conversion screen and analyzed with the ImageMaster 2DE Platinum software 5.0 (GE Healthcare, Piscataway, NJ). Briefly, all gels images were processed and analyzed under the same parameters and, after spot detection, automatically matched with the spots of a master gel used as a reference. Landmark spots were used to confirm spot matching across all gels and manual verification was used to screen out any dust artefacts or incorrectly identified spots. Relative volume of each spot in a gel was normalized as a percentage of the total volume of all spots detected on the gel. For statistical analysis, the spot volumes were imported into SAS software (SAS/STAT, 2011). A protein spot was considered statistically significant across breed and aging when p< 0.05.
ACCEPTED MANUSCRIPT 2.6. Protein identification by mass spectrometry Protein of interest from SDS-PAGE and 2DE preparative gels were destained, digested with trypsin
T
as reported by Giangrande et al. (2013) and analysed using a 6520 Accurate-Mass Q-TOF LC/MS
IP
System (Agilent Technologies, Palo Alto, CA, USA) equipped with a 1200 HPLC system and a
SC R
chip cube (Agilent Technologies). After loading, the peptide mixture was first concentrated and washed on 40 nL enrichment column (Agilent Technologies chip), with 0.1% formic acid in 2%
NU
acetonitrile as the eluent. The sample was then fractionated on a C18 reverse-phase capillary column (Agilent Technologies chip) at flow rate of 400 nL/min, with a linear gradient of eluent B
MA
(0.1% formic acid in 95% acetonitrile) in A (0.1% formic acid in 2% acetonitrile) from 7% to 80% in 50 min. Peptide analysis was performed using data-dependent acquisition of one MS scan (mass
D
range from 300 to 1,800 m/z) followed by MS/MS scans of the five most abundant ions in each MS
TE
scan. MS/MS spectra were measured automatically when the MS signal surpassed the threshold of 50,000 counts. The acquired MS/MS spectra were transformed in mz.data format and used for
CE P
protein identification with MASCOT 2.1 (Matrix Science, Boston, MA, USA). The Mascot search parameters were: trypsin as enzyme, allowed number of missed cleavage 3,
AC
carbamidomethyl as fixed modification, oxidation of methionine, pyro-Glu N-term Q as variable modifications, 10 ppm MS tolerance and 0.6 Da MS/MS tolerance and peptide charge from +2 to +3. Spectra with a MASCOT score of
S0309-1740(14)00155-7 doi: 10.1016/j.meatsci.2014.05.024 MESC 6431
To appear in:
Meat Science
Received date: Revised date: Accepted date:
30 July 2013 27 February 2014 30 May 2014
Please cite this article as: Marino, R., Albenzio, M., della Malva, A., Caroprese, M., Santillo, A. & Sevi, A., Changes in meat quality traits and sarcoplasmic proteins during aging in three different cattle breeds, Meat Science (2014), doi: 10.1016/j.meatsci.2014.05.024
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Changes in meat quality traits and sarcoplasmic proteins
SC R
IP
T
during aging in three different cattle breeds
MA
NU
R. Marino*, M. Albenzio, A. della Malva, M. Caroprese, A. Santillo, A. Sevi
Dipartimento di Scienze Agrarie, degli Alimenti e dell’Ambiente, University of Foggia, Via
TE
D
Napoli, 25, 71121, Foggia, Italy.
*Corresponding author: Tel.: +39 0881 589330; fax: +39 0881589502.
AC
CE P
Email address: [email protected] (R.Marino)
ACCEPTED MANUSCRIPT Abstract The effects of breed and aging time (1, 7, 14, 21d) were evaluated on physical meat properties and
T
on sarcoplasmic proteins changes in 24 young bulls from Romagnola x Podolian, Podolian and
IP
Friesian breed. Aging affects lightness showing an increase in all breeds while changes in redness
SC R
varied according to the breed. Podolian breed showed meat with the darkest and the reddest color and the lowest drip loss compared to the other breeds. Extending aging to 21d reduced drip loss
NU
from meat. SDS-PAGE and 2DE showed that many changes in the sarcoplasmic proteins occurred among breeds and during aging. During post-mortem some sarcoplasmic proteins decline in
MA
intensity after 21d highlighting that they were susceptible to aging. Proteins identification and western blotting showed the presence of myosin light chains, Troponin T and tropomyosin proteins
D
during aging, suggesting a degradation of myofibers and a more intense proteolysis especially in
CE P
TE
Podolian breed.
AC
Keywords: breed, aging, water holding capacity, sarcoplasmic proteins, proteolysis.
1. Introduction Eating satisfaction is one of the most important characteristics by which consumers judge meat quality after purchase (Grunert, Bredahl, & Brunso, 2004); tenderness, color and juiciness are the major components of beef palatability (Serra, Ruiz-Ramirez, Arnau, & Gou, 2005; Muchenje et al., 2009). Several studies (Ruiz de Huidobro, Miguel, Onega, & Blazquez, 2003; Revilla, & VivarQuintana, 2006) reported that these parameters are dependent on the rate and extent of post mortem aging. A number of factors contribute to post mortem tenderization of meat, in particular, many
ACCEPTED MANUSCRIPT authors (Koohmaraie, 1996; Taylor, Geesink, Thompson, Koohmaraie, & Goll, 1995; Kolczak, Pospiech, Palka, & Lacki, 2003; Huff-Lonergan, Zhang, & Lonergan, 2010) indicate that
T
proteolytic degradation of myofibrillar proteins plays an important role in tenderization showing
IP
ultrastructural changes in skeletal muscle.
SC R
Besides to myofibrillar proteins, sarcoplasmic fraction is the other compound of muscle proteins that account for 30-35% of total proteins and is primarily represented by glycolitic enzymes and
NU
myoglobin. Previous research (Ohlendieck, 2010) reported that the alteration of the sarcoplasmic fraction is not directly involved in the muscle tenderness, because the sarcoplasmic proteins have
MA
non structural functions and they are soluble in situ. Even though, studies in pig reported that the denaturation of sarcoplasmic proteins has an impact on meat quality parameters such as color and
D
water holding capacity (Sayd et al., 2006; Joo, Kauffman, Kim, & Park, 1999).
TE
Consumers’ choices are concerned not only about meat quality but also its origin (Grunert et al.,
CE P
2004). In particular, the use of rustic or autochthonous breed has the advantage that these animals are closely related to the environment and maintain the biodiversity (Revilla et al., 2006; Cuvelier et al., 2006). A large number of genetically distinct cattle breed are reared in Italy and this genetic
AC
diversity produces meat with many peculiar features. The effect of breed and aging on meat tenderness and on proteolytic pattern of myofibrillar proteins are reported in a previous study (Marino et al., 2013), data showed that meat tenderness and proteolysis were different among breeds with different purpose. To our knowledge, limited data are available on changes in meat sarcoplasmic proteins among different cattle breeds during aging. Therefore, in the present study we investigated the effect of breed and aging time on physical meat properties (pH, color, water holding capacity) and related sarcoplasmic proteins changes in longissimus dorsi muscle in three breeds with different productive purpose.
2. Materials and methods
ACCEPTED MANUSCRIPT 2.1. Animals and meat sampling Twenty four young bulls of three cattle breeds with different productive purpose, dairy (Friesian),
T
beef (Romagnola x Podolian crossbreed) and rustic (Podolian), were used in this study, details on
IP
breed characteristics are described in Marino et al. (2013). Animals (8 for each breed) were reared
SC R
intensively during the finishing period and were slaughtered at 19 months of age, according to industrial routines used in Italy and to the EU rule n. 119/1993.
NU
24 h post mortem Longissimus dorsi (LD) muscle was removed from each half of the carcass. Each muscle was divided longitudinally into two sections resulting in four samples for each animal.
MA
Then, each sample was stored at 2°C under vacuum packaging and analyzed at 1, 7, 14 and 21 d of aging, respectively. Cranial and caudal sections were randomized across aging time. pH, color
D
parameters, Warner Bratzler shear force (WBS), water holding capacity and changes in
TE
sarcoplasmic proteins were estimated. The effects of aging and breed on WBS are reported in a
CE P
previous paper (Marino et al., 2013).
AC
2.2. pH measurement, color measurements and water holding capacity (WHC) Ten g of each sample was blended with 90 ml of peptone water in a Stomacher bag for 30 s using a Stomacher (Interscience, France), the pH of the mixture was measured using a pH meter (Crison Instrument, Barcelona, Spain). Color was measured using a color meter Minolta CR 400 (Konica Minolta, Osaka, Japan) on 1 cm thick steaks after storage at 3 ± 1 °C for 1 h. Results were expressed as L* (lightness), a* (redness) and b* (yellowness) according to the standard conditions of the Commission International d’Eclairage (CIE). The values were measured in five location on the surface of each slice after 1, 7, 14 and 21 days.
ACCEPTED MANUSCRIPT Water holding capacity was measured as thawing loss. 1 cm thick frozen steak from each sample was placed on plastic netting over a polystyrene tray and stored in a plastic bag for 48 h at 4°C.
T
After storage, the meat was removed from the tray and the weight of the tray plus the juice was
SC R
thawing loss (%) = [(Wt + j − Wt)/ (Wi + m − Wt)] × 100,
IP
recorded. Thawing loss was expressed as a percentage of the initial weight of the meat:
where Wt is the weight of the empty tray, Wt + j is the weight of the tray plus the juice, and Wi + m
2.3. Extraction of sarcoplasmic proteins
MA
NU
is the weight of the tray with meat.
D
Samples were freed of connective and adipose tissue, after 2.5 g were homogenized with an Ultra
TE
Turrax homogenizer (IKA T18 basic, Germany) with 10 ml of 0.003M Phosphate Buffer at pH 7,
CE P
for 3 min. The homogenate was centrifuged under refrigeration at 4°C and 8000 x g (Eppendorf 5810 R, Eppendorf AG, Hamburg, Germany) for 20 min. After centrifugation the supernatant was aliquotated and frozen at -80°C. Protein concentrations were determined by the Bradford protein
AC
assay (Bio-Rad Laboratories, Hercules, CA) using bovine serum albumin (BSA) as a standard.
2.4 Gel sample preparation and SDS-PAGE Analysis Sarcoplasmic proteins were resolved by Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in a gradient gel 8-18%. The run was performed in a continuous buffer system using a Protean II xi vertical slab gel unit (Bio-Rad Laboratories, Hercules, CA). Gels were stained with Coomassie blue G250. Destained gel images were acquired by the Chemi Doc EQ system (Bio-Rad Laboratories, Hercules, CA) using a white light conversion screen and analyzed with the Quantity One software (Bio-Rad Laboratories, Hercules, CA) to determine the signal intensity (optical intensity) of the defined bands. Identification of the protein molecular weight was done by
ACCEPTED MANUSCRIPT comparison with a known molecular weight standard (precision plus protein standard-broad range, Bio-Rad, Laboratories, Hercules, CA). With the sum of the intensity of the defined bands in a lane
T
set a 100%, the relative quantity of each band was determined as the percentage of the signal
2.5. Two dimensional Gel Electrophoresis (2DE)
SC R
IP
intensity of the defined band in a lane.
NU
Protein separation in the first dimension was made in immobilized pH gradient (IPG) dry strips (17 cm IPG strips, Bio-Rad Laboratories, Hercules, CA), spanning the pH regions 3-10 for the
MA
sarcoplasmic extract using an IPG Protean IEF Cell (Bio-Rad Laboratories, Hercules, CA). The sarcoplasmic extract was suspended in isoelectrofocusing IPG sample buffer (ready-Prep
D
Rehydratation/Sample Buffer, Bio-Rad Laboratories, Hercules, CA). The IPG strips were
TE
equilibrated at room temperature for 15 min in equilibration buffers I and II (Bio-Rad Laboratories, Hercules, CA). Two dimensional separation was performed on a Protean II xi vertical slab gel unit
CE P
(Bio-Rad Laboratories, Hercules, CA) using SDS-PAGE gradient 8–18%. A broad range molecular weight electrophoresis calibration kit (Bio-Rad Laboratories, Hercules, CA) was used as standard.
AC
Gels were fixed and stained with Coomassie blue G250 (Bio-Rad Laboratories, Hercules, CA). The destained gels were acquired by the Chemi Doc EQ system (Bio-Rad Laboratories, Hercules, CA) using a white light conversion screen and analyzed with the ImageMaster 2DE Platinum software 5.0 (GE Healthcare, Piscataway, NJ). Briefly, all gels images were processed and analyzed under the same parameters and, after spot detection, automatically matched with the spots of a master gel used as a reference. Landmark spots were used to confirm spot matching across all gels and manual verification was used to screen out any dust artefacts or incorrectly identified spots. Relative volume of each spot in a gel was normalized as a percentage of the total volume of all spots detected on the gel. For statistical analysis, the spot volumes were imported into SAS software (SAS/STAT, 2011). A protein spot was considered statistically significant across breed and aging when p< 0.05.
ACCEPTED MANUSCRIPT 2.6. Protein identification by mass spectrometry Protein of interest from SDS-PAGE and 2DE preparative gels were destained, digested with trypsin
T
as reported by Giangrande et al. (2013) and analysed using a 6520 Accurate-Mass Q-TOF LC/MS
IP
System (Agilent Technologies, Palo Alto, CA, USA) equipped with a 1200 HPLC system and a
SC R
chip cube (Agilent Technologies). After loading, the peptide mixture was first concentrated and washed on 40 nL enrichment column (Agilent Technologies chip), with 0.1% formic acid in 2%
NU
acetonitrile as the eluent. The sample was then fractionated on a C18 reverse-phase capillary column (Agilent Technologies chip) at flow rate of 400 nL/min, with a linear gradient of eluent B
MA
(0.1% formic acid in 95% acetonitrile) in A (0.1% formic acid in 2% acetonitrile) from 7% to 80% in 50 min. Peptide analysis was performed using data-dependent acquisition of one MS scan (mass
D
range from 300 to 1,800 m/z) followed by MS/MS scans of the five most abundant ions in each MS
TE
scan. MS/MS spectra were measured automatically when the MS signal surpassed the threshold of 50,000 counts. The acquired MS/MS spectra were transformed in mz.data format and used for
CE P
protein identification with MASCOT 2.1 (Matrix Science, Boston, MA, USA). The Mascot search parameters were: trypsin as enzyme, allowed number of missed cleavage 3,
AC
carbamidomethyl as fixed modification, oxidation of methionine, pyro-Glu N-term Q as variable modifications, 10 ppm MS tolerance and 0.6 Da MS/MS tolerance and peptide charge from +2 to +3. Spectra with a MASCOT score of
