Rapid determination of amino acid incorporation by stable isotope labeling with amino acids in cell...

RAPID COMMUNICATIONS IN MASS SPECTROMETRY

Rapid Commun. Mass Spectrom. 2007; 21: 3919–3926

) DOI: 10.1002/rcm.3290
Published online in Wiley InterScience (www.interscience.wiley.com
Rapid determination of amino acid incorporation

by stable isotope labeling with amino acids in

cell culture (SILAC)

Frank Schmidt, Margarita Strozynski, Sandra S. Salus, Hilde Nilsen and Bernd Thiede*The Biotechnology Centre of Oslo, University of Oslo, Gaustadalleen 21, 0349 Oslo, Norway

Received 24 May 2007; Revised 13 September 2007; Accepted 1 October 2007

*CorrespoUniversit0317 OsloE-mail: b

Stable isotope labeling with amino acids in cell culture (SILAC) has evolved to be a major technique

for quantitative proteomics using cell cultures. We developed a rapid method to follow and

determine the incorporation of arginine and lysine. Analysis of the heavy state is required to avoid

quantification errors. Moreover, the mixture of light and heavy states can be exploited to normalize

the protein amount for subsequent relative quantification experiments. Therefore, peptides from

different cell lines were extracted with 0.1% trifluoroacetic acid and analyzed bymatrix-assisted laser

desorption/ionization tandem time-of-flight (MALDI-TOF/TOF) mass spectrometry (MS). This

analysis was highly reproducible and was performed in less than 2h, significantly faster than other

methods for the same purpose. Similar peptide mass profiles were obtained for human EBV-

transformed B, Jurkat T, and HeLa cells as well as for mouse embryonic fibroblasts. Proteolytic

fragments of 27 human proteins were identifiedwith 56 peptides byMALDI-MS/MS and can be used

as a database for these kinds of experiments. Sequencing revealed that the peptides were predo-

minantly amino- and carboxy-terminal protein fragments displaying a specificity characteristic of the

acidic proteases cathepsin D and E.Many of the identified peptides contained arginine and/or lysine,

allowing determination of the incorporation rate of these amino acids. Furthermore, the rate of con-

version of arginine into proline could bemonitored easily. Copyright# 2007 JohnWiley& Sons, Ltd.

Mass spectrometry (MS)-based methods that utilize stable

isotopes for quantitative proteome analyses have undergone

significant development in recent years.1 Stable isotope label-

ing with amino acids in cell culture (SILAC) is a simple and

straightforward approach for in vivo incorporation of a label

into proteins for quantitative proteomics by mass spectro-

metry.2 Consequently, SILAC has been applied to many

quantitative proteomics analyses, including functional and

clinical studies.3–21 SILAC involves culturing of one cell

population in normalmedium (light) and another inmedium

containing specific heavy isotopic amino acids, making their

respective peptides distinguishable by mass. These cell

populations can then be subjected to different treatments and

combined prior to further processing. During analysis byMS,

the relative peak intensities between the unlabeled and

labeled peptides are quantitative, making it possible to

evaluate the effects of specific treatment on a large number of

proteins within a single experiment.

The majority of proteins are typically unchanged during

an experiment. To find regulated proteins using stable

isotopes, it is generally recommended to normalize the

relative protein input from controls and sample of interest to

a 1:1 ratio. However, the accuracy is unsatisfactory for

ndence to: B. Thiede, The Biotechnology Centre of Oslo,y of Oslo, Gaustadalleen 21, P.O. Box 1125 Blindern,, [email protected]

protein quantification according to Bradford22 or Lowry23

assay. Analysis of tryptic peptides by MS can be performed

for relative quantificationwith orwithout prior separation by

sodium dodecyl sulfate polyacrylamide gel electrophoresis

(SDS-PAGE) of mixed proteins labeled with light and heavy

amino acids, but this method is relatively slow and requires

approximately 2 days.24 Furthermore, SILAC experiments

depend on readout by MS. Thus, it must be ensured that

proteins are fully labeled to avoid quantification errors.24

For SILAC experiments, relative quantification is required

for normalization of the protein amount of the light and

heavy state. Furthermore, full incorporation of heavy amino

acids is required to exclude quantification errors. In this

study, a simple and rapid methodology, based on peptide

analysis with matrix-assisted laser desorption/ionization

(MALDI)-MS of different cell lines, was established to follow

and determine the incorporation of amino acids by SILAC.

We found that this approach allowed identification and

relative quantification of a number of peptides that were

derived from larger proteins in less than 2h. Further-

more, the rate of conversion of arginine into proline could be

monitored easily. Notably, the determined proteolytic

fragments can be useful as a database for these kinds of

experiments.

Copyright # 2007 John Wiley & Sons, Ltd.

3920 F. Schmidt et al.

EXPERIMENTAL

Isolation of peptidesThe Jurkat T cell line E6, EBV-transformed human B-cells,

HeLa cells, and mouse embryonic fibroblasts were main-

tained in RPMI tissue culture medium without arginine and

lysine (Invitrogen, Karlsruhe, Germany) supplemented

with 10% dialyzed fetal calf serum (Invitrogen), penicillin

(100U/mL)/streptomycin (100mg/mL) (Invitrogen) at 378Cin 5.0% CO2, and 100mg/L of arginine-12C6 monohydro-

chloride and lysine-12C6 monohydrochloride (light) or

arginine-13C6 monohydrochloride and lysine-13C6 monohydro-

chloride (heavy) (Sigma-Aldrich, Oslo, Norway). Only

50mg/L arginine monohydrochloride was used for the

experiments with reduced arginine. The cells were washed

twice with phosphate-buffered saline (PBS) at room tem-

perature (RT). Subsequently, 1� 106 cells were treated with

100mL of 0.1% trifluoroacetic acid (TFA) for 5min at 48C.Then, the cells were homogenized with a pellet pestle

(Sigma-Aldrich), incubated for 5min and centrifuged for

10min at 10 000 rpm. The supernatant was purified using

m-C18 ZipTips (Millipore, Eschborn, Germany) and eluted

directly onto a MALDI sample plate with 0.4mL a-cyano-

4-hydroxycinnamic acid (CHCA, Bruker Daltonics, Bremen,

Germany; 5mg/mL in 0.2% aqueous TFA/acetonitrile (3:2)).

Liquid chromatographyThe purified and dried peptides were dissolved in 5mL

sample solution (0.1% TFA in water) and loaded onto

an UltiMateTM 3000 nanoLC (Dionex, Idstein, Germany)

equipped with a FLM 3X00 (integrated flow manager) and

micro-vacuum degasser. Peptide separation was performed

on a C18 PepMap 100 column (3.0mm, 150mm� 75mm) for

gradient elution (eluent A: 0.05% TFA in water and 5%

acetonitrile; eluent B: 0.05% TFA in 95% acetonitrile) from

0 to 50% eluent B within 40min and for further 5min from

50% to 90% eluent B. After 15min, the eluted sample

fractions were continuously diluted with 0.5mL/min CHCA

and spotted onto a MALDI target using a Probot (LC-

Packings/Dionex, California, USA) with an interval of 1min

resulting in 30 fractions.

MALDI-TOF/TOF MSThe mass spectra were recorded with the software FlexCon-

trol 2.4 (Bruker Daltonics, Bremen, Germany) using a

MALDI-tandem time-of-flight (TOF/TOF) mass spectro-

meter (Ultraflex II, Bruker Daltonics, Bremen, Germany).

External calibration was performed with the peptides kemp-

tide, bradykinin, substance P free acid, glu-fibrinopeptide B,

and dynorphin A (Sigma-Aldrich, Steinheim, Germany). The

peak lists were created from the raw data by FlexAnalysis 2.4

(Bruker Daltonics, Bremen, Germany) using mass-range

700–4000Da (MS), and minimal signal-to-noise (S/N) ratio 6

(MS) as peak filter settings. MS/MS spectra of pre-selected

peaks were generated manually. The MS/MS peptide

identifications were achieved by database comparisons

(SwissProt database, 20050621, human, 12359 sequences)

using Biotools 3.0 (Bruker Daltonics, Bremen, Germany)

in conjunction with the Mascot in-house version 2.0.0.25 A

tolerance of 50 ppm was used for the precursor mass and


0.4Da for MS/MS fragments. Moreover, taxonomy human,

none enzyme, andN-acetyl (protein) as variablemodification

were selected. Peptides were considered to be identified

when the probability based individual ion scores were at

least 40with rank 1. For relative quantification, the intensities

of labeled and unlabeled peptides with a shift of 6.02Da for

arginine and lysine and 5.02Da for proline-containing

peptides (as a side product of the arginine biosynthesis)

were determined with the program FlexAnalysis 2.4 (Bruker

Daltonics, Bremen, Germany).

RESULTS

Peptide mass profiling of differentcell lines by MALDI-MSThe objective was to establish a fast and sensitive procedure

to determine the incorporation of arginine and lysine in

peptides from cells cultured in SILAC medium. After SILAC

treatment, 1� 106 cells were incubated with 100mL 0.1% TFA

for 5min, homogenized, and centrifuged. The peptides in the

supernatant were directly purified and concentrated with

m-C18 ZipTips, spotted onto aMALDI target and the peptide

mass profile was recorded. Representative mass spectra of

Jurkat T cells, HeLa cells, EBV-transformed human B cells,

and mouse embryonic fibroblasts (MEF) are shown in Fig. 1.

The peptide mass profiles of the three human cell lines were

comparable, although differences in intensity of the detected

masses were observed. The mouse cell line showed similar

peptide mass profiles in comparison to the human cells.

However, a peptide with significant intensity at m/z 2092.98

was detected in MEF cells, whereas a peptide was missing

which was detected with high intensity at m/z 2076.97 in

human cells. MS/MS analysis of both peptides confirmed the

known sequence variation of glyceraldehyde-3-phosphate

dehydrogenase from phenylalanine in human to tyrosine in

mouse within the same sequence. The most prominent

mass peaks of the peptide mass profiles were subjected to

fragmentation by MALDI-MS/MS in order to identify the

proteins these peptides were derived from (Fig. 1). All

peptides were fragments of proteins, predominantly derived

from, or close to, the amino- or carboxy-termini (Fig. 1,

Table 1). The most intense mass peaks corresponded

to 10 kDa heat shock protein (CH10; m/z 1551.84, and

1397.71), glyceraldehyde-3-phosphate dehydrogenase (G3P2;

m/z 2077.01, 1690.81, and 1169.61), macrophage migration

inhibitory factor (MIF; m/z 1785.98), peptidyl-prolyl cis-trans

isomerase A (PPIA; m/z 1716.93, 1864.00, 1474.78), actin

(ACTB; m/z 2409.23, 2223.13), GTP-binding nuclear protein

Ran (RAN; m/z 1372.78), and nucleophosmin (NPM;

m/z 1003.57). In addition, fragmentation patterns with

polyglutamic acid stretches were detected for the mass

peaks at m/z 1101.42 and 1230.46, which could correspond to

different proteins.

Peptide mass profiling of JurkatT cells by LC/MALDI-MS/MSThe peptides derived from Jurkat T cells were further

separated by nano-liquid chromatography (LC). The LC

fractions were analyzed by MALDI-MS to detect the

same peptides as in the peptide mass profiles. In summary,


DOI: 10.1002/rcm

Figure 1. Peptide mass profiles of different cell lines. Peptide mass profiles of Jurkat T cells (A), HeLa cells (B),

EBV-transformed human B cells (C), and mouse embryonic mouse cells (D) are shown. Proteins that give rise to

peptides causing themajor mass peaks are shownwith their human Swiss-Prot entry name. The significant mass peak

at m/z 2092.98 in MEF cells (D) corresponds to glyceraldehyde-3-phosphate dehydrogenase of mouse

(G3P_MOUSE).

Copyright # 2007 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2007; 21: 3919–3926

DOI: 10.1002/rcm

Rapid determination of SILAC incorporation 3921

Table 1. Proteins identified by peptide mass profiling of human cells. The human entry names, protein names, and accession

numbers according to Swiss-Prot/TrEmbl are displayed. Amino- and carboxy-terminal peptides are highlighted in gray at protein

range. Proteins identified bymanual de novo sequencing are depicted at Mascot score and the identified amino acids are shown at

peptide sequence as bold letters. Furthermore, two different sequenceswithin the same protein entry were found for actin (#), most

likely due to an exchange of cysteine to tryptophan. Furthermore, the mitochondrial precursor of 60 kDa heat shock protein was

deleted from the sequence to define the protein range (�)

Entry name Protein name

Accession

no.

Mascot

score

Peptide

mass

Protein

range Peptide sequence P1/P1́ P1/P1́

ACTB Actin, cytoplasmic 1 P60709 82 2223.09 357–375(375) ISKQEYDESGPSIVHRKCF WI

de novo 2306.16 357–375(375)# ISKQEYDESGPSIVHRKWF WI

96 2409.17 356–375(375) WISKQEYDESGPSIVHRKCF MW

ALDOA Fructose-bisphosphate aldolase A P04075 de novo 837.39 357–363(363) FVSNHAY LF

40 1655.90 63–78(363) LTADDRVNPCIGGVIL LF LF

BACH Cytosolic acyl coenzyme A

thioester hydrolase

O00154 57 1963.98 310–327(380) VDADPVVDSSQKRYRAAS LV SA

CH10 10 kDa heat shock protein, mitochondrial P61604 41 1176.64 1–10(101) Ac-AGQAFRKFLP PL

57 1289.73 1–11(101) Ac-AGQAFRKFLPL LF

73 1397.71 90–101(101) FRDGDILGKYVD LF

61 1551.82 1–13(101) Ac-AGQAFRKFLPLFD DR

CH60 60 kDa heat shock protein, mitochondrial P10809 50 2118.14 1–20(547)� AKDVKFGADARALMLQGVDL LL

89 2926.33 518–547(547)� VVVTEIPKEEKDPGMGAMGGMGGGMGGGMF EV

COF1 Cofilin-1 P23528 95 1819.86 84–98(165) YDATYETKESKKEDL LY LV

DUT Deoxyuridine 5́-triphosphate

nucleotidohydrolase, mitochondrial

P33316 54 1995.94 233–252(252) VQALDDTERGSGGFGSTGKN EV

EF1A1 Elongation factor 1-alpha 1 P68104 40 1198.59 415–424(462) FSDYPPLGRF SF FA

ENOA Enolase alpha P06733 75 1764.87 283–297(443) IKDYPVVSIEDPFDQ FI QD

46 2159.11 24–44(433) FTSKGLFRAAVPSGASTGIYE LF EA

63 2324.23 1–20(433) Ac-SILKIHAREIFDSRGNPTVE EV

53 2651.41 1–23(433) Ac-SILKIHAREIFDSRGNPTVEVDL LF

G3P2 Glyceraldehyde-3-phosphate P04406 60 1264.52 313–322(334) YDNEFGYSNR WY RV

dehydrogenase, liver 69 1577.69 313–325(334) YDNEFGYSNRVVD WY DL

57 1586.79 77–90(334) QERDPSKIKWGDAG FQ GA

52 1690.77 313–326(334) YDNEFGYSNRVVDL WY LM

47 1949.94 77–93(334) QERDPSKIKWGDAGAEY FQ YV

78 1963.88 310–325(334) ISWYDNEFGYSNRVVD LI DL

76 2076.97 310–326(334) ISWYDNEFGYSNRVVDL LI LM

H2AL Histone H2A.1 Q93077 90 1801.08 114–129(129) VLLPKKTESHHKAKGK AV

H2AX Histone H2A.x P16104 46 2650.53 114–140(142) VLLPKKTSATVGPKAPSGGKKATQASQ AV QE

IPYR Inorganic pyrophosphatase Q15181 57 1427.63 1–13(278) Ac-SGFSTEERAAPFS SL

KPYM Pyruvate kinase isozymes M1/M2 P14618 de novo 2093.03 1–19(530) Ac-SKPHSEAGTAFIQTQQLHA AA

de novo 2295.11 1–21(530) Ac-SKPHSEAGTAFIQTQQLHAAM MA

METK S-adenosylmethionine synthetase gamma P31153 71 1406.61 1–12(386) Ac-MNGQLNGFHEAF FI

MIF Macrophage migration inhibitory factor P14174 66 1438.77 4–17(114) IVNTNVPRASVPDG FI GF

52 1585.83 3–17(114) FIVNTNVPRASVPDG MF GF

53 1785.95 4–20(114) IVNTNVPRASVPDGFLS FI SE

47 1933.02 3–20(114) FIVNTNVPRASVPDGFLS MF SE

44 2028.08 3–22(114) IVNTNVPRASVPDGFLSEL FI LT

79 2489.32 23–45(114) TQQLAQATGKPPQYIAVHVVPDQ LT QL

NPM Nucleophosmin P06748 48 1003.55 288–294(294) WQWRKSL LW

PAIRB Plasminogen activator inhibitor 1

RNA-binding protein

Q8NC51 54 2003.94 1–18(392) PGHLQEGFGCVVTNRFDQ QL

PPIA Peptidyl-prolyl cis-trans isomerase A P62937 63 1141.59 8–18(164) DIAVDGEPLGR FD RV

78 1288.65 7–18(164) FDIAVDGEPLGR FF RV

41 1716.88 8–23(164) DIAVDGEPLGRVSFEL FD LF

42 1863.95 7–23(164) FDIAVDGEPLGRVSFEL FF LF

PROF1 Profilin-1 P07737 90 1988.08 40–58(139) VNITPAEVGVLVGKDRSSF FV FY

142 2759.51 32–58(139) AAVPGKTFVNITPAEVGVLVGKDRSSF WA FY

PSA7 Proteasome subunit alpha type 7 O14818 52 1994.95 1–18(248) Ac-SYDRAITVFSPDGHLFQ QV

RAN GTP-binding nuclear protein Ran P62826 58 1357.70 1–12(205) Ac-AAQGEPQVQFKL LV

48 1372.76 165–176(205) ARKLIGDPNLEF LA FV

RLA2 60S acidic ribosomal protein P2 P05387 40 1115.59 1–9(115) MRYVASYLL LA

ROA1 Heterogeneous nuclear P09651 40 1147.53 30–38(371) RSHFEQWGT LR TL

ribonucleoprotein A1 43 2172.16 1–19(371) Ac-SKSESPKEPEQLRKLFIGG GL

RS17 40S ribosomal protein S17 P08708 79 1957.03 117–134(134) QVTQPTVGMNFKTPRGPV LQ

SODC Superoxide dismutase P00441 145 2072.11 1–20(153) Ac-ATKAVCVLKGDGPVQGIINF FE

STMN1 Stathmin P16949 73 2106.07 1–19(148) Ac-ASSDIQVKELEKRASGQAF FE

65 2348.20 1–21(148) Ac-ASSDIQVKELEKRASGQAFEL LI

Copyright # 2007 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2007; 21: 3919–3926

DOI: 10.1002/rcm



56 peptides of 27 proteins could be identified by MALDI-

MS/MS (Table 1). Eighteen amino-terminal sequences,

including 15 acetylated forms, 10 carboxy-terminal sequen-

ces, and 14 sequences originating less than ten amino acids

from the terminal ends, were detected (Table 1). Several

proteins yielded more than one peptide, usually with

overlapping sequences. Peptides from the amino- and the

carboxy-terminal ends were identified for the 10 kDa and

Figure 2. Peptide mass profiles of Jurkat Tcells. Peptide mas

heavy-13C6 (B); arginines and/or lysines are shown. In additi

detected peptides contained arginines and lysines that can be

mixture (C).


60 kDa heat shock proteins (Table 1). Cleavage tended to

occur predominantly at hydrophobic amino acid residues.

Leucine (35 times), phenylalanine (34 times), valine (15 times),

alanine (12 times) and isoleucine (10 times) were the most

frequent amino acids N-terminal to the cleavage site, but

valine and isoleucine were not found at position P1.

Furthermore, tryptophan (8 times) was found relatively

often, considering the low occurrence of this amino acid in

s profiles of Jurkat Tcells cultured with light-12C6 (A) and

on, the 1:1 mixture is depicted (C). Notably, most of the

seen by the occurrence of numerous mass pairs in the


DOI: 10.1002/rcm


the human proteome. On the other hand, asparagine,

cysteine, histidine, and lysine were never observed to be

involved in cleavage and proline was found only once.

The observed cleavage specificity is typical for the acidic

proteases cathepsin D and E.26 Accordingly, the addition of

pepstatin A to the 0.1% TFA solution, a known inhibitor of

acidic proteases, abolished the production of the peptides.

Rapid determination of arginineand lysine incorporation by SILACPeptide mass profiling was employed to determine the

amino acid incorporation rate during cell culture. Compari-

son of the peptide mass profiles of Jurkat T cells cultured

with light-12C6 (Fig. 2(a)) and heavy-13C6 (Fig. 2(b)) arginine

and lysine showed that the most abundant peptides

displayed mass differences of 6, 12, and 18Da. These mass

differences were in accordance with the number of arginines

and lysines within the peptide sequences (Table 1). The lack

of peptides with light arginines and lysines in the peptide

mass profile of Jurkat T cells cultured with heavy amino

acids confirmed full incorporation of the amino acids and

was observed for all detected peptides (Fig. 2(b)). Moreover,

the ratio of light to heavy (L/H) can be determined bymixing

the samples. A 1:1 mixture of 106 cells from both light and

heavy cultures revealed a peptide mass profile with more

Figure 3. Peptide mass pairs of a 1:1 mixture of Jurkat T cell

Six peptide mass pairs corresponding to six different proteins w

(L/H) ratio was calculated by the intensities of the peptide pairs.

mass peaks at þ5Da to the heavy peptides were considered f


than 10 mass pairs with high intensity and S/N ratio

(Fig. 2(c)). The L/H ratio of the individual mass pairs was on

average 1.02� 0.07 (Figs. 2(c) and 3). Interestingly, full

incorporation of heavy amino acids in Jurkat T and HeLa

cells was observed for all identified proteins as early as two

cell splittings (96 h) after exposure of the SILAC medium

(Fig. 4). However, the slower growing MEF cells required

four cell splittings for full incorporation of arginine and

lysine.

Rapid determination of conversionof 13C6-arginine into 13C5-prolineSome cell lines convert 13C6-arginine into

13C5-proline, which

results in the formation of two distinct peak clusters for all of

the labeled proline-containing peptides.27 The correct peak

intensity of the heavy state is therefore the sum of both the13C6-arginine and the 13C5-proline mass peaks. Actually,

some of the identified peptides contained prolines (Table 1).

Considering the four cell lines used, significant additional

mass peaks that were þ5Da compared to the peptide with

heavy arginines and/or lysines were observed only in Jurkat

T cells (Fig. 3). The conversion of 13C6-arginine into13C5-proline

was observed as early as one round of sub-culture post-SILAC

addition, which corresponded to 48 h of SILAC exposure

(Figs. 4(a) and 4(b)). The conversion increased only slightly,

s cultured with either 12C6- or13C6-arginines and lysines.

ere selected from those shown in Fig. 2. The light to heavy

Due to the formation of 13C5-proline, the intensities of the

or the calculation of the ratios.


DOI: 10.1002/rcm

Figure 4. Peptide mass profiling to monitor arginine and lysine incorporation efficiency and conversion of proline. Full

incorporation of arginine and lysine was observed in Jurkat T cells after the second round of sub-culture because the light

form (m/z 1551.83) of the peptide Ac-AGQAFRKFLPLFD of the mitochondrial 10 kDa heat shock protein was only detected

after one cell splitting (A, B). The formation of 13C5-proline detected atm/z 1568.89 increased only marginally between split 2

and 8 (A). Halving the amount of added arginine slightly decreased the conversion of 13C6-arginine into 13C5-proline while

preserving the incorporation efficiency of arginine.


exemplified for the most abundant mass m/z 1563.86, from

15% after two and 19% after eight rounds of sub-culture

(Fig. 4(a)). Similar values were observed for other proline-

containing peptides (Fig. 3). The conversion of 13C6-arginine

into 13C5-proline could be reduced to some extent using

50mg/L 13C6-arginine in RPMI medium (Fig. 4(b)) instead

of 100mg/L (Fig. 4(a)). Notably, the same amount of12C6-arginine must be used for relative quantification

experiments.

DISCUSSION

The incorporation of amino acids by SILACmust be checked

for every cell line. The cell population of the heavy state

must be analyzed to determine that the heavy amino acids

are incorporated and that the light amino acids are not

detectable. This analysis usually requires up to 2 days.24 In

this study, we exploited protein cleavage products of

different human cell lines to follow and determine the


incorporation rate of amino acids by SILAC within less than

2 h. Similar peptide mass profiles were obtained from four

different cell lines and revealed a number of well-known

abundant proteins. This suggests that the peptide mass list

presented here (Table 1) could be of general use. Interest-

ingly, the protein fragments revealed that cleavage was

mainly due to cathepsin D and E activity and most peptides

were derived from, or close to, either the amino- or

carboxy-termini. Significantly, the observed peptide mass

profile contained fragments derived from several proteins

and was not dominated by one or two extremely abundant

proteins. Hence, we have developed a simple, direct, and

rapid mass spectrometric approach to determine the

incorporation and interconversion of amino acids by SILAC.

Our approach is significantly faster than other methods to

determine and normalize the relative quantities of light and

heavy labeled isotopes. Moreover, it can be used to rapidly

monitor whether full incorporation of labeled amino acids by

SILAC has occurred such that the samples can be employed

in subsequent biological experiments.


DOI: 10.1002/rcm


REFERENCES

1. Ong SE, Mann M. Nat. Chem. Biol. 2005; 1: 252.2. Ong SE, Blagoev B, Kratchmarova I, Kristensen DB, Steen H,

Pandey A, Mann M. Mol. Cell. Proteomics 2002; 1: 376.3. Olsen JV, Blagoev B, Gnad F, Macek B, Kumar C, Mortensen

P, Mann M. Cell 2006; 127: 635.4. Bose R, Molina H, Patterson AS, Bitok JK, Periaswamy B,

Bader JS, Pandey A, Cole PA. Proc. Natl. Acad. Sci. USA 2006;103: 9773.

5. Cantin GT, Venable JD, Cociorva D, Yates JR III. J. ProteomeRes. 2006; 5: 127.

6. Gronborg M, Kristiansen TZ, Iwahori A, Chang R, Reddy R,Sato N, Molina H, Jensen ON, Hruban RH, Goggins MG,Maitra A, Pandey A. Mol. Cell. Proteomics 2006; 5: 157.

7. Hwang SI, Lundgren DH, Mayya V, Rezaul K, Cowan AE,Eng JK, Han DK. Mol. Cell. Proteomics 2006; 5: 1131.

8. Liang X, Zhao J, HajivandiM,WuR, Tao J, Amshey JW, PopeRM. J. Proteome Res. 2006; 5: 2632.

9. Selbach M, Mann M. Nat. Methods 2006; 3: 981.10. Thiede B, Kretschmer A, Rudel T. Proteomics 2006; 6: 614.11. Vinther J, Hedegaard MM, Gardner PP, Andersen JS, Arc-

tander P. Nucleic Acids Res. 2006; 34: e107.12. Amanchy R, Kalume DE, Iwahori A, Zhong J, Pandey A.

J. Proteome Res. 2005; 4: 1661.13. Andersen JS, Lam YW, Leung AK, Ong SE, Lyon CE,

Lamond AI, Mann M. Nature 2005; 433: 77.


14. Gruhler A, Olsen JV, Mohammed S, Mortensen P, Faerge-man NJ, Mann M, Jensen ON. Mol. Cell. Proteomics 2005;4: 310.

15. Romijn EP, Christis C, Wieffer M, Gouw JW, Fullaondo A,van der Sluijs P, Braakman I, Heck AJ. Mol. Cell. Proteomics2005; 4: 1297.

16. Blagoev B, Ong SE, Kratchmarova I, MannM.Nat. Biotechnol.2004; 22: 1139.

17. de Hoog CL, Foster LJ, Mann M. Cell 2004; 117: 649.18. Everley PA, Krijgsveld J, Zetter BR, Gygi SP. Mol. Cell.

Proteomics 2004; 3: 729.19. Ibarrola N, Molina H, Iwahori A, Pandey A. J. Biol. Chem.

2004; 279: 15805.20. Ong SE, Mittler G, Mann M. Nat. Methods 2004; 1:

119.21. Blagoev B, Kratchmarova I, Ong SE, Nielsen M, Foster LJ,

Mann M. Nat. Biotechnol. 2003; 21: 315.22. Bradford MM. Anal. Biochem. 1976; 72: 248.23. Lowry OH, Rosbrough NJ, Farr AL, Randall RJ. J. Biol. Chem.

1951; 193: 265.24. Ong SE, Mann M. Nat. Protoc. 2006; 1: 2650.25. Perkins DN, Pappin DJ, Creasy DM, Cottrell JS. Electropho-

resis 1999; 20: 3551.26. Arnold D, Keilholz W, Schild H, Dumrese T, Stevanovic S,

Rammensee HG. Eur. J. Biochem. 1997; 249: 171.27. Ong SE, Kratchmarova I, Mann M. J. Proteome Res. 2003;

2: 173.


DOI: 10.1002/rcm

Rapid determination of amino acid incorporation by stable isotope labeling with amino acids in cell...

Documents

Transcript of Rapid determination of amino acid incorporation by stable isotope labeling with amino acids in cell...