Coupling of High Resolution CE and LC to MALDI MS  

INTRODUCTION

Our laboratory has been involved in coupling of high resolution LC and CE to MALDI MS since 1997. We have developed LC-MALDI interfaces for applications requiring both on-line and off-line coupling .  This page reviews of several  interfaces we have developed.

MALDI MS is well-established for mass analysis of large molecules, and has been highly effective in protein identification from, for example, in-gel digests of 2D gel electrophoresis of complex protein mixtures.  Since the introduction of interfaces coupling MALDI to liquid phase separations several years ago, LC-MALDI MS (or CE-MALDI-MS) has demonstrated complementary advantages to the earlier-established LC-ESI-MS methods.   Our early interfaces were, like ESI-MS, coupled in an on-line mode, where the eluent is mixed with the MALDI matrix and transferred directly to the source chamber of the mass spectrometer.  Subsequent work took advantage of the opportunity to perform LC-MS in an off-line mode, in which the eluent is deposited onto a standard MALDI plate and subsequently analyzed in the mass spectrometer:

  1. Sample separation and deposition are decoupled from the MS analysis, thus avoiding the constraints of on-line data dependent acquisition.
  2. An entire separation can be rapidly surveyed in the MS mode first ,to determine which peaks need to be analyzed in a second round of MS/MS analysis.  
  3. Inclusion and exclusion lists for MS/MS acquisition can be easily modified during the MS/MS acquisition, interactively with database searching results.
  4. Separation is deposited and archived on a solid surface; select samples can be reanalyzed weeks or months later.
  5. Multiplex LC separations can be deposited in parallel on a single MALDI plate, multiplying LC-MS throughput.  

 

ON-LINE DEPOSITION INTERFACE

The separated sample is mixed with MALDI matrix in liquid junction and directly transfered into the vacuum chamber of the mass spec where it is deposited on a polymer tape (Mylar) via a tapered capillary [Ref 1,2].  The deposited sample is then transported by cassette recorder like mechanism to the repeller where MALDI process takes place.  The interface was introduced in 1998 for coupling with capillary electrophoresis. In Ref. 2 it was shown that separation efficiencies as high as 300,000 theoretical plates can be preserved using this interface.  In 2001 the interface was upgraded for operation in the multiplex mode with 8 CE separations carried out in parallel.

On-line CE

OFF-LINE DEPOSITION INTERFACE


Off-line deposition interface CE

UNIVERSAL DEPOSITION INTERFACE

Allows deposition in both continuous mode and spotting mode. See table below for more info.

Continuous mode: LC eluent mixed with MALDI matrix is deposited via capillary that is touching the MALDI plate under app. 60 degree angle in evacuated chamber (200 Torr). The MALDI plate is coated with thin layer of nitrocellulose prior to deposition. The lower pressure in the chamber and nitrocellulose coating ensure formation of continuous streak of sample.  The pressure in the chamber controls the evaporation of the solvent and can be adjusted to accommodate flow rates ranging from app. 200 nL/min to 3 uL/min.  The continuous deposition allows retaining of separation resolution even for chromatographic peaks as narrow as 1-2 sec (FWHM) and collection of full MS spectra every 0.1 sec of chromatographic separation. Primary applications for continuous deposition are capillary electrophoresis, monolithic HPLC separations, etc.  More discussion about the influence of number of data points per chromatographic peak can be found here.

Spotting mode: LC eluent mixed with MALDI matrix is deposited on MALDI plate in the form of spots under atmospheric pressure. This configuration is advantageous for less demanding separation such as coupling of standard nano and capillary LC separations to MALDI MS.  The interface allows spotting at a rate of up to app. 2-3 sec per chromatographic data point.

Off-lineLC

Characteristics of the Universal Deposition Interface

Universal interface SEM photo of the trace
SEM photos courtesy of Dr. P. Savickas, Applied Biosystems




APPLICATIONS

Closely Placed External Mass Calibrants


Multiplexing Deposition

We developed multiplex interface using on-line 8-channel capillary electrophoresis in 2000 [Ref]. Though the multiplex operation allowed significantly higher sample throughput, the interface was compatible only with our lab-built instrument without MS/MS capabilities.  At the ASMS 2002 meeting we have introduced another multiplex system using off-line continuous deposition coupled to nano LC. 
Multiplexing with single injector

Upgraded version of multiplex system was also presented at the ASMS 2004 meeting.  In order to overcome the rather slow serial sample introduction, we have implemented individual sample injectors for each separation channel.

Multiplexing with individual injectors

APPLICATIONS:



Monolithic LC Column


MALDI MS AND MS/MS ANALYSIS

The samples are deposited on a standard MALDI plates (Applied Biosystems 2" x 2" format) for analysis using:

MORE INFO

REFERENCES


Last update July 2004, Tomas