How to make nano-frit

http://www.abrf.org/index.cfm/list.msg/abrf/73705

Date: Mon, 10 Mar 2008 09:08:48 -0400
From: "J. Will Thompson"
To: "'ABRF Discussion List'"
Subject: RE: nano column frits

Hi Chris and Brian,

I have also used this material with a slightly different approach to make high-quality nanocolumn frits. Our technique uses Kasil 2130 (also available free in 1 liter), formamide, and a glass wool fiber filter. Make a clean cut on the end of the capillary and place the fiber filter on a hard surface like a lab benchtop. Place 20uL of Kasil 2130 on the glass filter. Pipet 20ul of formamide directly on top of the Kasil. Using gloves so you can grip the capillary well, depress the end of the capillary into the glass filter and rotate back and forth between thumb and forefinger. Lift and depress in a different spot on the wetted filter; repeat 15-20 times. Each time the capillary will make a small "hole" in the filter and the result should be a 0.5 to 1 mm frit in the end of the capillary (no need to trim the end of the capillary afterwards). Heat at 100C for 30 min to cure. I have used this to frit capillaries from 30 um to 150 um in diameter.
One note, the Kasil/formamide mix cures very quickly so use one spot on a filter paper per capillary you wish to frit. Since you are only using 20ul at a time, the free sample should give you about 50,000 capillary frits.

Good luck!

Will

J. Will Thompson, Ph.D.
Proteomics Core Facility
B02 Levine Science Research Center
Institute for Genome Sciences & Policy
Duke University Medical Center
will.thompson@duke.edu

Consumables from Bruker Daltonics On-line at http://tinyurl.com/79nmaub

I have look around for list of consumables from Bruker Daltonics. Perhaps it was my faults for not looking hard enough, but for my records, I keep it here at http://tinyurl.com/79nmaub.

In particular, below is bacteria test standards for MALDI Biotyper information and preparation protocols.
http://tinyurl.com/82bgem7


The highly sophisticated Bruker Bacterial Test Standard (BTS) contains a typical extract of Escherichia coli DH5alpha. In a MALDI-TOF mass spectrum, this extract shows a characteristic peptide and protein profile. Moreover, the extract is spiked with two additional proteins. Thus, the BTS covers an overall mass range from 3.6 to 17 kDa. Each tube contains material for the preparation of about 40 spots.


To prepare for use,
0. Prepare 50:47.5:2.5 H2O:ACN:TFA solution ("standard solvent").
1. Add 50 μl of standard solvent to the pellet and dissolve by pipetting up and down for at least 20 times at room temperature. Avoid foaming of the solution!
2. Incubate the standard for 5 min at room temperature and repeat pipetting up and down for at least 20 times.
3. Centrifuge for 2 min at room temperature (13000 rpm).
4. Prepare aliquots containing 5 μl of dissolved BTS. For this purpose, use micro tubes connected with screw caps (see above).
5. Use only one micro tube for sample preparation. Store the remaining aliquots at -18°C or below.

Direct comparison between automated biochemical technique vs MALDI-TOF based microorganism ID

Comparison between automated method and MALDI-TOF for the microbial identification is show clearly that MALDI is unbiased and accurate.

http://onlinelibrary.wiley.com/doi/10.1111/j.1469-0691.2010.03328.x/abstract;jsessionid=71A6CC99DA640BD80748EA58073939A5.d01t01

Combining with other crucial benefits, such as speed, simplicity, cost-effective, no consumables required, MALDI-TOF based microbial ID is a necessity in every microbiology lab.

Mascot Error at BIOTEC

The mascot database used at BIOTEC for years gave error complaining that it could not map memory for NCBInr.

Remove to check marks at the top of the database maintenance and configuration.

Also
IgnoreDuplicate EST_Other NCBInr

In-gel digestion protocol

=========================
In-gel digestion Protocol
=========================

1. Digest 3 BSA gel plugs and analyze with Ultraflex TOF/TOF with correct identifcation for all three. Repeat if fail.

2. Digest one BSA gel plug as control with the digestion of samples.

=========
Protocol
=========
1. Place gel plug in a 96 well plate.

•    1 pc/well for 2D gel-plug;
•    5 pcs of 1x1x1 mm3/well for coomassie;
•    10 pcs 1x1x1 mm3/well for silver stained.

2. Add 200 uL of sterile water shake for 5 min at room temperature.

3. Remove water and add 200 uL of 100% ACN. Shake for 5 min at room temperature.

4. Remove ACN and allow the gel plug to dry at room temperature for 5-10 min.

5. Add 50 uL 10mM dithiothreitol/10mM ammonium bicarbonate/well. Allow to sit at room temperature for reduction of disulfide bond for 1 hr.

6. Remove 10mM Dithiothreitol/10mM ammonium bicarbonate.

7. Add 50 uL 100mM Iodoacetamide/10mM ammonium bicarbonate. Keep in the dark place at room temperature for 1 hr.

8. Remove 100 mM Iodoacetamide/10mM ammonium bicarbonate.

9. Add 200 uL of 100% ACN. Shake at room temperature for 5 min. Remove ACN. Repeate this step twice. (Total 3 times). Repeat once more if the gel has not shrunk.

10. Add 20uL of 10ng Trypsin in 50% ACN/10mM ammonium bicarbonate (for coomassie, use 40uL). Leave at room temperature for 20 min before adding 30 uL of 30% ACN and leave at room temperature for 3 hrs.

11. Transfer liquid to another 96 well plate. (If the well is dry, add 30 uL of 30% ACN, shake at RT before transfering.

12. Extract the rest of peptide in 11 by with 30 uL 50% ACN/0.1% FA, and shake for at RT for 10 min. Transfer to the same plate in 10. Repeat twice.

13. Dry in a 40 degree incubator for 3-4 hr or overnight. A heat block or speed vac can be used.

14. Keep sample at -80C until analysis.

==========================================
Reconsitute sample in 5uL of 0.1 % FA.
Spot on MALDI plate with mixing ratio 1:1.
==========================================

=======================
Preparation of Reagents
=======================

20 mM ammonium biocarbonate (FW 79.06) 50 mL.
Weigh 79.06 mg of ammonium bicarbonate. Dissolve in milliQ water adjust volume to 50 mL.

10 mM ammonium bicarbonate 50 mL
Add 25 mL of 20 mM NH4HCOO to a 50 mL volumetric flask. Adjust volume with water to 50 mL.

10 mM dithiothreitol/10mM ammonium bicarbonate 2 mL (freshly prepare).
Weigh DTT (FW 154.25) 3.085 mg dissolve in 10 mM ammonium bicarbonate adjust volume to 2 mL.

100 mM Iodoacetamide / 10 mM ammonium bicarbonate 2mL.
Weight Iodoacetamide (FW 184) 36.8 mg dissolve in 10 mM ammonium bicarbonate.

50% acetonitrile/10 mM ammonium bicarbonate
Add 100% ACN to 20mM ammonium bicarbonate 1:1

10ng trypsin in 50% ACN/10mM ammonium bicarbonate (for silverstain).
Add 2 mL of 50% ACN/10mM ammonium bicarbonate to 20ug of trypsin.

30% ACN 5mL (used 1.92 mL for 96 well).
Add 1.5 mL of 100% ACN to 3.5 mL milliQ water.

0.1% FA 50 mL
Add 50 uL of concentrate FA to 49.95 mL of milliQ water.

50% ACN in 0.1% FA 10 mL (8.64 mL for sample 96 well)
Add 100% ACN 5 mL in 0.1%FA mL.

Publication List

Publication List:

1. Clipston, NL; Jai-nhuknan, J; Cassady, CJ A comparison of negative and positive ion time-of-flight post-source decay mass spectrometry for peptides containing basic residues INTERNATIONAL JOURNAL OF MASS SPECTROMETRY 2003, 363-381

2. Puapaiboon, U; Jai-nhuknan, J; Cowan, JA Characterization of a multi-functional metal-mediated nuclease by MALDI-TOF mass spectrometry NUCLEIC ACIDS RESEARCH 2001, 3652-3656

3. Puapaiboon, U; Jai-nhuknan, J; Cowan, JA Rapid and direct sequencing of double-stranded DNA using exonuclease III and MALDI-TOF MS ANALYTICAL CHEMISTRY 2000, 3338-3341

4. Puapaiboon, U; Jai-nhuknan, J; Cowan, JA Rapid and direct sequencing of double-stranded DNA using exonuclease III and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). BIOCHEMISTRY 2000, 64

5. Puapaiboon, U; Taylor, RT; Jai-nhuknan, J Structural confirmation of polyurethane dendritic wedges and dendrimers using post source decay matrix-assisted laser desorption/ionization time-of-flight mass spectrometry RAPID COMMUNICATIONS IN MASS SPECTROMETRY 1999, 516-520

6. Chan, KW; Power, TD; Jai-nhuknan, J; Cybulski, SM An ab initio study of He-F-2, Ne-F-2, and Ar-F-2 van der Waals complexes JOURNAL OF CHEMICAL PHYSICS 1999, 860-869

7. Jai-nhuknan, J; Cassady, CJ Negative ion postsource decay time-of-flight mass spectrometry of peptides containing acidic amino acid residues ANALYTICAL CHEMISTRY 1998, 5122-5128

8. Jai-nhuknan, J; Cassady, CJ Negative ion matrix-assisted laser desorption/ionization time-of-flight post-source decay calibration by using fibrinopeptide B JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY 1998, 540-544

9. Zhang, X;Jai-Nhuknan, J; Cassady, CJ Collision-induced dissociation and post-source decay of model dodecapeptide ions containing lysine and glycine INTERNATIONAL JOURNAL OF MASS SPECTROMETRY 1997, 135-145

http://www.ms-utils.org/wiki/pmwiki.php/Main/SoftwareList

http://www.ms-utils.org/wiki/pmwiki.php/Main/SoftwareList

internal standard for peptide digest

Add 10 uL of glu-fib 500 fmol/uL for 100 uL of sample (at 100fmol/uL). Works fine.

start easy-nanosprayer on maXis

1. Replace the normal spacer with "Low Flow spacer".
2. Check the tip of the sprayer if the core capillary is fully retracted in the steel tube.
3. Insert the nanospray assembly on to the ESI source.
4. Turn the gold wheel clockwise to extend the core capillary (can't observe).
5. Connect syringe pump with tune mix (L ESI-TOF version) flow at 60 uL/h)
6. Adjust nebulizer as required.

The easy-nanospray needs to be primed with 1uL/min flow of mobile phase or tune mix solution. I prefer the latter, as I can check and recalibrate the system at the same time. At 1 uL/min flow, nebulizer was set as low as 0.1 bar to achieve the most intense signal.

Integration of Shimadzu Prominence HPLC to HyStar

1. Back up system
2. Connect Network Card #1 to CBM-20A Lite (with or without Ethernet Switch)
3. Setup a static IP address 10.104.52.20 for Network card #1
4. Install the Shimadzu-HyStar plug-in
5. In HyStar's hardware setup, choose Shimadzu HPLC as the LC System
6. Click LC System Settings
7. Choose CBM-20A Lite as controller, click "configure"
7. Specify IP address of 10.104.52.14 for CBM-20A Lite Connection
8. Configure other devices (except Detector--which should be left blank)
9. Click Save
10. In HyStar hardware setup, click close.