Lidstrom:13C Incorporation Into Protein - Data Analysis - Revision history

Janet B. Matsen: /* Our protocol */

2012-12-27T00:23:38Z

Our protocol

←Older revision		Revision as of 00:23, 27 December 2012
Line 27:		Line 27:
	** serine is a good 1st check: comes from 3PG		** serine is a good 1st check: comes from 3PG
	** Alanine is a good 2nd to check: comes from pyruvate. (?? why not valine, leucine, or isoleucine??)		** Alanine is a good 2nd to check: comes from pyruvate. (?? why not valine, leucine, or isoleucine??)
-	[[image:amino acids from central metabolism.jpg\|thumb\|upright=1.0\|center\|central metabolism: sources of amino acids]]	+	*[[image:amino acids from central metabolism.jpg\|thumb\|upright=1.0\|center\|central metabolism: sources of amino acids]]
	** don't do methionine b/c formate can be unintentionally incorporated as formyl-methionine (for ATG codon)		** don't do methionine b/c formate can be unintentionally incorporated as formyl-methionine (for ATG codon)
	** don't use the following: [http://www.ncbi.nlm.nih.gov/pubmed/17822305 source:] Accurate assessment of amino acid mass isotopomer distributions for metabolic flux analysis, 2007. (A paper Yanfen recommended)		** don't use the following: [http://www.ncbi.nlm.nih.gov/pubmed/17822305 source:] Accurate assessment of amino acid mass isotopomer distributions for metabolic flux analysis, 2007. (A paper Yanfen recommended)

Janet B. Matsen: /* Our protocol */

2012-12-27T00:22:59Z

Our protocol

←Older revision		Revision as of 00:22, 27 December 2012
Line 29:		Line 29:
	[[image:amino acids from central metabolism.jpg\|thumb\|upright=1.0\|center\|central metabolism: sources of amino acids]]		[[image:amino acids from central metabolism.jpg\|thumb\|upright=1.0\|center\|central metabolism: sources of amino acids]]
	** don't do methionine b/c formate can be unintentionally incorporated as formyl-methionine (for ATG codon)		** don't do methionine b/c formate can be unintentionally incorporated as formyl-methionine (for ATG codon)
-	** don't use the following:	+	** don't use the following: [http://www.ncbi.nlm.nih.gov/pubmed/17822305 source:] Accurate assessment of amino acid mass isotopomer distributions for metabolic flux analysis, 2007. (A paper Yanfen recommended)
	***cysteine and tryptophan: can get oxidized during hydrolysis		***cysteine and tryptophan: can get oxidized during hydrolysis
	***glutamine and asparagine: can be deamidated to glutamate and aspartate, respectively		***glutamine and asparagine: can be deamidated to glutamate and aspartate, respectively

Janet B. Matsen: /* Our protocol */

2012-12-27T00:20:17Z

Our protocol

←Older revision		Revision as of 00:20, 27 December 2012
Line 25:		Line 25:

	* You can chose which amino acid(s) to look at:		* You can chose which amino acid(s) to look at:
-	** serine is a good 1st check	+	** serine is a good 1st check: comes from 3PG
-	** Alanine is a good 2nd to check	+	** Alanine is a good 2nd to check: comes from pyruvate. (?? why not valine, leucine, or isoleucine??)
	[[image:amino acids from central metabolism.jpg\|thumb\|upright=1.0\|center\|central metabolism: sources of amino acids]]		[[image:amino acids from central metabolism.jpg\|thumb\|upright=1.0\|center\|central metabolism: sources of amino acids]]
	** don't do methionine b/c formate can be unintentionally incorporated as formyl-methionine (for ATG codon)		** don't do methionine b/c formate can be unintentionally incorporated as formyl-methionine (for ATG codon)
		+	** don't use the following:
		+	***cysteine and tryptophan: can get oxidized during hydrolysis
		+	***glutamine and asparagine: can be deamidated to glutamate and aspartate, respectively
		+	***histidine derivative: less stable, may degrade.

	==Data Analysis from Martin==		==Data Analysis from Martin==

Janet B. Matsen: /* Our protocol */

2012-12-27T00:07:42Z

Our protocol

←Older revision		Revision as of 00:07, 27 December 2012
Line 27:		Line 27:
	** serine is a good 1st check		** serine is a good 1st check
	** Alanine is a good 2nd to check		** Alanine is a good 2nd to check
-	[[image:amino acids from central metabolism.jpg\|thumb\|upright=3.0\|center\|central metabolism: sources of amino acids]]	+	[[image:amino acids from central metabolism.jpg\|thumb\|upright=1.0\|center\|central metabolism: sources of amino acids]]
	** don't do methionine b/c formate can be unintentionally incorporated as formyl-methionine (for ATG codon)		** don't do methionine b/c formate can be unintentionally incorporated as formyl-methionine (for ATG codon)

Janet B. Matsen: /* Our protocol */

2012-12-27T00:03:16Z

Our protocol

←Older revision		Revision as of 00:03, 27 December 2012
Line 23:		Line 23:
	## Example: If looking for Serine M-57, the mass of the unique ion should be 390.		## Example: If looking for Serine M-57, the mass of the unique ion should be 390.
	# If you want to go back to full chromatogram, Chromatogram → Redraw TIC. (TIC = total ion chromatogram)		# If you want to go back to full chromatogram, Chromatogram → Redraw TIC. (TIC = total ion chromatogram)
		+
		+	* You can chose which amino acid(s) to look at:
		+	** serine is a good 1st check
		+	** Alanine is a good 2nd to check
		+	[[image:amino acids from central metabolism.jpg\|thumb\|upright=3.0\|center\|central metabolism: sources of amino acids]]
		+	** don't do methionine b/c formate can be unintentionally incorporated as formyl-methionine (for ATG codon)

	==Data Analysis from Martin==		==Data Analysis from Martin==

Janet B. Matsen: /* Our protocol */

2012-12-20T23:11:22Z

Our protocol

←Older revision		Revision as of 23:11, 20 December 2012
Line 4:		Line 4:
	# Open the program: Start --> Programs --> MSD ChemStation --> Instrument #1 --> Instrument #1 Data Analysis		# Open the program: Start --> Programs --> MSD ChemStation --> Instrument #1 --> Instrument #1 Data Analysis
	# Load the compound library from Z:\Common\Software\ChemStation Library/NIST08.L		# Load the compound library from Z:\Common\Software\ChemStation Library/NIST08.L
-	# Load a sample file folder in navigator to the left.	+	# Load a sample file folder in navigator to the left. (right click)
	# Chose an ion to look for		# Chose an ion to look for
	## Chose an amino acid, the use Yanfen's table to see the unique ion retention time and m/z.		## Chose an amino acid, the use Yanfen's table to see the unique ion retention time and m/z.

Janet B. Matsen: /* Our protocol */

2012-12-20T23:00:16Z

Our protocol

←Older revision		Revision as of 23:00, 20 December 2012
Line 2:		Line 2:

	== Our protocol ==		== Our protocol ==
-	# Open Start --> Programs --> MSD ChemStation --> Instrument #1 --> Instrument #1 Data Analysis	+	# Open the program: Start --> Programs --> MSD ChemStation --> Instrument #1 --> Instrument #1 Data Analysis
	# Load the compound library from Z:\Common\Software\ChemStation Library/NIST08.L		# Load the compound library from Z:\Common\Software\ChemStation Library/NIST08.L
		+	# Load a sample file folder in navigator to the left.
	# Chose an ion to look for		# Chose an ion to look for
	## Chose an amino acid, the use Yanfen's table to see the unique ion retention time and m/z.		## Chose an amino acid, the use Yanfen's table to see the unique ion retention time and m/z.
Line 10:		Line 11:
	# Chromatogram --> extract ion chromatogram		# Chromatogram --> extract ion chromatogram
	## Enter time range that includes your expected retention time and a few minutes of buffer.		## Enter time range that includes your expected retention time and a few minutes of buffer.
		+	## Enter the m/z for the unique ion (use M-57)
		+	# Make sure you have the right compound:
		+	## double right click in the peak area. Double right click on the spectrum to match it to the library.
		+	# Subtract background
		+	##double right click on an area that is not the peak. Spectrum → subtract.
		+	# Get Ion Intensities
		+	##use right clicker to draw a rectangle with vertical edges at the ½ maximal height
		+	# Spectrum → tabulate
		+	## gives intensities for all ions in the time interval specified by your rectangle.
		+	# Look for 13C shifts
		+	## Example: If looking for Serine M-57, the mass of the unique ion should be 390.
		+	# If you want to go back to full chromatogram, Chromatogram → Redraw TIC. (TIC = total ion chromatogram)

	==Data Analysis from Martin==		==Data Analysis from Martin==

Janet B. Matsen: /* Our protocol */

2012-12-20T22:54:22Z

Our protocol

←Older revision		Revision as of 22:54, 20 December 2012
Line 8:		Line 8:
	### serine is a good 1st choice.		### serine is a good 1st choice.
	### after serine, look at ____.		### after serine, look at ____.
		+	# Chromatogram --> extract ion chromatogram
		+	## Enter time range that includes your expected retention time and a few minutes of buffer.

	==Data Analysis from Martin==		==Data Analysis from Martin==

Janet B. Matsen: /* Our protocol */

2012-12-20T22:49:18Z

Our protocol

←Older revision		Revision as of 22:49, 20 December 2012
Line 5:		Line 5:
	# Load the compound library from Z:\Common\Software\ChemStation Library/NIST08.L		# Load the compound library from Z:\Common\Software\ChemStation Library/NIST08.L
	# Chose an ion to look for		# Chose an ion to look for
-	## ~~Use~~ Yanfen's table. serine is a good 1st choice.	+	## Chose an amino acid, the use Yanfen's table to see the unique ion retention time and m/z.
-	## ~~After~~ serine, look at ____.	+	### serine is a good 1st choice.
		+	### after serine, look at ____.

	==Data Analysis from Martin==		==Data Analysis from Martin==

Janet B. Matsen: /* Our protocol */

2012-12-20T22:48:38Z

Our protocol

←Older revision		Revision as of 22:48, 20 December 2012
Line 4:		Line 4:
	# Open Start --> Programs --> MSD ChemStation --> Instrument #1 --> Instrument #1 Data Analysis		# Open Start --> Programs --> MSD ChemStation --> Instrument #1 --> Instrument #1 Data Analysis
	# Load the compound library from Z:\Common\Software\ChemStation Library/NIST08.L		# Load the compound library from Z:\Common\Software\ChemStation Library/NIST08.L
-	#	+	# Chose an ion to look for
		+	## Use Yanfen's table. serine is a good 1st choice.
		+	## After serine, look at ____.

	==Data Analysis from Martin==		==Data Analysis from Martin==